Cardiac sarcoplasmic reticulum. Effects of an atherogenic diet during the neonatal and juvenile period

Energy Technology Data Exchange (ETDEWEB)

Jacobson, M S; Ambudkar, I S; Young, E P; Naseem, S M; Heald, F P; Shamoo, A E [Maryland Univ., College Park (USA). School of Medicine

1985-04-01

The effect on the cardiac sarcoplasmic reticulum of an atherogenic (1% cholesterol) diet fed during the neonatal vs the juvenile period of life was studied in Yorkshire swine. Male piglets were randomly assigned at birth to 1 of 4 groups: group I (control), group II (lactation feeding), group III (juvenile period feeding) and group IV (lactation and juvenile feeding). All animals were killed at 55 weeks of age and cardiac sarcoplasmic reticulum (SR) isolated for assay of calcium uptake, Ca/sup 2 +/-Mg/sup 2 +/ ATPase activity, and lipid analysis by thin-layer chromatography and gas chromatography. The amount of cholesterol/mg SR protein and the cholesterol/phospholipid ratio were higher in the animals fed during lactation (groups II and IV) and lower in those fed only during the juvenile period (group III). Phospholipid fatty acid patterns as measured by gas chromatography were unaltered in any group. Calcium uptake was markedly diminished in all experimental conditions: group II 47%, group III 65% and group IV 96%. Compared to the observed changes in calcium transport, the ATP hydrolytic activity was relatively less affected. Only in group IV a significant decrease (41%) was seen. Groups II and III show no change in ATP hydrolytic activity. The decrease in calcium uptake and altered cholesterol/phospholipid ratio without effect on ATP hydrolytic activity is consistent with an uncoupling of calcium transport related to the atherogenic diet in early life.

Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

Science.gov (United States)

de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

1995-05-01

Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

Energy Technology Data Exchange (ETDEWEB)

Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

1978-01-01

Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca²⁺ + Mg²⁺ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca²⁺ + Mg²⁺ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca²⁺ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

International Nuclear Information System (INIS)

Campbell, K.P.

1978-01-01

Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca 2+ + Mg 2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca 2+ + Mg 2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca 2+ /mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization

Specific protein-protein interactions of calsequestrin with junctional sarcoplasmic reticulum of skeletal muscle

International Nuclear Information System (INIS)

Damiani, E.; Margreth, A.

1990-01-01

Minor protein components of triads and of sarcoplasmic reticulum (SR) terminal cisternae (TC), i.e. 47 and 37 kDa peptides and 31-30 kDa and 26-25 kDa peptide doublets, were identified from their ability to bind 125 I calsequestrin (CS) in the presence of EGTA. The CS-binding peptides are specifically associated with the junctional membrane of TC, since they could not be detected in junctional transverse tubules and in longitudinal SR fragments. The 31-30 kDa peptide doublet, exclusively, did not bind CS in the presence of Ca 2+ . Thus, different types of protein-protein interactions appear to be involved in selective binding of CS to junctional TC

The influence of ionizing radiation on the structure of Ca-ATPase hydrophobic fragment of skeletal muscle sarcoplasmic reticulum

International Nuclear Information System (INIS)

Vojtsitskij, V.M.; Fedorov, A.N.; Lugovskoj, Eh.B.; Derzskaya, S.G.; Khizhnyak, S.V.; Kurskij, M.D.; Kucherenko, N.E.

1990-01-01

Early (1 and 24 h) after X-irradiation with a dose of 0.21 C/kg changes occurred in the acceptibility of the polypeptide chain parts of sarcoplasmic reticulum Ca-ATPase for the effect of trypsin. The analysis of the results of studying the structural and functional properties of a hydrophobic fragment of this enzyme in the control and after irradiation permitted to define the part of the Ca-ATPase polypeptide chain that provided ion selectivity of the fragment

Vanilloid receptor expressed in the sarcoplasmic reticulum of rat skeletal muscle

International Nuclear Information System (INIS)

Xin Hong; Tanaka, Hideyuki; Yamaguchi, Maki; Takemori, Shigeru; Nakamura, Akio; Kohama, Kazuhiro

2005-01-01

Vanilloid receptor subtype 1 (VR1) was cloned as a capsaicin receptor from neuronal cells of dorsal root ganglia. VR1 was subsequently found in a few non-neuronal tissues, including skeletal muscle [Onozawa et al., Tissue distribution of capsaicin receptor in the various organs of rats, Proc. Jpn. Acad. Ser. B 76 (2000) 68-72]. We confirmed the expression of VR1 in muscle cells using the RT-PCR method and Western blot analysis. Immunostaining studies with a confocal microscope and an electron microscope indicated that VR1 was present in the sarcoplasmic reticulum (SR), a store of Ca 2+ . The SR releases Ca 2+ to cause a contraction when a muscle is excited. However, SR still releases a small amount of Ca 2+ under relaxed conditions. We found that this leakage was enhanced by capsaicin and was antagonized by capsazepine, a capsaicin blocker, indicating that leakage of Ca 2+ occurs through a channel composed of VR1

Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

Science.gov (United States)

Tadini-Buoninsegni, Francesco; Moncelli, Maria Rosa; Peruzzi, Niccolò; Ninham, Barry W.; Dei, Luigi; Nostro, Pierandrea Lo

2015-10-01

The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

Specific binding of [3H]LY186126, an analogue of indolidan (LY195115), to cardiac membranes enriched in sarcoplasmic reticulum vesicles

International Nuclear Information System (INIS)

Kauffman, R.F.; Utterback, B.G.; Robertson, D.W.

1989-01-01

LY186126 was found to be a potent inhibitor of type IV cyclic AMP phosphodiesterase located in the sarcoplasmic reticulum of canine cardiac muscle. This compound, a close structural analogue of indolidan (LY195115), was prepared in high specific activity, tritiated form to study the positive inotropic receptor(s) for cardiotonic phosphodiesterase inhibitors such as indolidan and milrinone. A high-affinity binding site for [ 3 H]LY186126 was observed (Kd = 4 nM) in purified preparations of canine cardiac sarcoplasmic reticulum vesicles. Binding was proportional to vesicle protein, was inactivated by subjecting membranes to proteolysis or boiling, and was dependent on added Mg2+. Scatchard analysis suggested the presence of a single class of binding sites in the membrane preparation. Indolidan, milrinone, and LY186126 (all at 1 microM) produced essentially complete displacement of bound [ 3 H]LY186126, while nifedipine, propranolol, and prazosin had little or no effect at this concentration. This represents the first reported use of a radioactive analogue to label the inotropic receptor for cardiotonic phosphodiesterase inhibitors. The results suggest that [ 3 H]LY186126 is a useful radioligand for examining the subcellular site(s) responsible for positive inotropic effects of these drugs

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

International Nuclear Information System (INIS)

King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

2006-01-01

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

Characterization of sarcoplasmic reticulum Ca(2+) ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy.

Science.gov (United States)

Guglielmi, V; Oosterhof, A; Voermans, N C; Cardani, R; Molenaar, J P; van Kuppevelt, T H; Meola, G; van Engelen, B G; Tomelleri, G; Vattemi, G

2016-06-01

Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

Science.gov (United States)

Lüdi, H; Hasselbach, W

1985-11-21

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

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SANDOR GYÖRKE

2004-01-01

Full Text Available Calsequestrin (CASQ2 is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR. Mutations in the cardiac calsequestrin gene (CASQ2 have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca2+-induced Ca2+ release (CICR and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.

Specific binding of (/sup 3/H)LY186126, an analogue of indolidan (LY195115), to cardiac membranes enriched in sarcoplasmic reticulum vesicles

Energy Technology Data Exchange (ETDEWEB)

Kauffman, R.F.; Utterback, B.G.; Robertson, D.W.

1989-05-01

LY186126 was found to be a potent inhibitor of type IV cyclic AMP phosphodiesterase located in the sarcoplasmic reticulum of canine cardiac muscle. This compound, a close structural analogue of indolidan (LY195115), was prepared in high specific activity, tritiated form to study the positive inotropic receptor(s) for cardiotonic phosphodiesterase inhibitors such as indolidan and milrinone. A high-affinity binding site for (/sup 3/H)LY186126 was observed (Kd = 4 nM) in purified preparations of canine cardiac sarcoplasmic reticulum vesicles. Binding was proportional to vesicle protein, was inactivated by subjecting membranes to proteolysis or boiling, and was dependent on added Mg2+. Scatchard analysis suggested the presence of a single class of binding sites in the membrane preparation. Indolidan, milrinone, and LY186126 (all at 1 microM) produced essentially complete displacement of bound (/sup 3/H)LY186126, while nifedipine, propranolol, and prazosin had little or no effect at this concentration. This represents the first reported use of a radioactive analogue to label the inotropic receptor for cardiotonic phosphodiesterase inhibitors. The results suggest that (/sup 3/H)LY186126 is a useful radioligand for examining the subcellular site(s) responsible for positive inotropic effects of these drugs.

Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle

International Nuclear Information System (INIS)

Bowman, B.J.; Berenski, C.J.; Jung, C.Y.

1985-01-01

Using radiation inactivation, the authors have measured the size of the H + -ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60 Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H + -ATPase in situ was found to be approximately 2.3 X 10(5) daltons. They also used radiation inactivation to measure the size of the Ca 2+ -ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, they directly compared the sizes of these two ATPases and found them to be essentially the same. The authors conclude that both H + -ATPase and Ca 2+ -ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides

Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle

DEFF Research Database (Denmark)

Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs

2000-01-01

during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P ... to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which......The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate...

Calcium uptake by sarcoplasmic reticulum isolated from hearts of septic rats

International Nuclear Information System (INIS)

McDonough, K.H.

1988-01-01

Myocardial sarcoplasmic reticulum (SR) plays a critical role in the regulation of the cytosolic calcium fluctuations that occur during the cardiac cycle. One function of the SR is to lower the calcium concentration so that myocardial relaxation and thus ventricular filling can occur. The aim of the present study was to determine if hyperdynamic sepsis induced a decrease in the capacity of SR to take up calcium. This defect would result in decreased ventricular filling and thus decreased cardiac output, as has previously been shown in isolated perfused working hearts removed from septic rats. Therefore, rats were anesthetized with ether, and sepsis was induced by the injection of an aliquot of a fecal homogenate into the peritoneal cavity. Control animals either underwent surgery and received an aliquot of sterilized fecal inoculum (sham) or were untreated (no surgery). On day 2 after surgery, animals were anesthetized with pentobarbital, and hearts were removed, weighted, and SR isolated. The rate of uptake of 45 Ca 2+ by SR from septic rats was not depressed compared to controls but in fact was elevated. Maximum 45 Ca 2+ accumulated by the SR and Ca 2+ -stimulated ATPase activity were similar in SR from control and septic hearts. These results suggest that the contractile dysfunction noted in the myocardium in early sepsis is probably not due to inadequate SR removal of Ca 2+ during diastole

Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

DEFF Research Database (Denmark)

Mahmmoud, Yasser Ahmed; Gaster, Michel

2013-01-01

BACKGROUND AND PURPOSE: The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling...... agents. EXPERIMENTAL APPROACH: Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. KEY RESULTS: Studies on SR vesicles showed that the endogenous......, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS: NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel...

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

Science.gov (United States)

Curran, Jerry; Tang, Lifei; Roof, Steve R; Velmurugan, Sathya; Millard, Ashley; Shonts, Stephen; Wang, Honglan; Santiago, Demetrio; Ahmad, Usama; Perryman, Matthew; Bers, Donald M; Mohler, Peter J; Ziolo, Mark T; Shannon, Thomas R

2014-01-01

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is

Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

DEFF Research Database (Denmark)

Nielsen, J S; Sahlin, K; Ørtenblad, N

2007-01-01

AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity....... METHODS: Isolated rat soleus muscles performed 30 EC bouts. Single fibres were isolated from the muscle and after mechanical removal of sarcolemma used to measure eSRCa2+, rate of SR Ca2+ loading and myofibrillar Ca2+ sensitivity. RESULTS: Following EC maximal force in whole muscle was reduced by 30......% and 16/100 Hz force ratio by 33%. The eSRCa2+ in fibres from non-stimulated muscles was 45 +/- 5% of the maximal loading capacity. After EC, eSRCa2+ per fibre CSA decreased by 38% (P = 0.05), and the maximal capacity of SR Ca2+ loading was depressed by 32%. There were no effects of EC on either...

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

OpenAIRE

Ishii, T; Takeyasu, K

1993-01-01

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity,...

Nandrolone decanoate treatment affects sarcoplasmic reticulum Ca(2+) ATPase function in skinned rat slow- and fast-twitch fibres.

Science.gov (United States)

Bouhlel, Aicha; Joumaa, Wissam H; Léoty, Claude

2003-09-01

The effects of anabolic-androgenic steroid administration on the function of the sarcoplasmic reticulum (SR) pump were investigated in chemically skinned fibres from the extensor digitorum longus (EDL) and soleus muscles of sedentary rats. Twenty male rats were divided into two groups, one group received an intramuscular injection of nandrolone decanoate (15 mg x kg(-1)) weekly for 8 weeks, the second received similar weekly doses of vehicle (sterile peanut oil). Compared with control muscles, nandrolone decanoate treatment reduced SR Ca(2+) loading in EDL and soleus fibres by 49% and 29%, respectively. In control and treated muscles, the rate of Ca(2+) leakage depended on the quantity of Ca(2+) loaded. Furthermore, for similar SR Ca(2+) contents, the Ca(2+) leakage rate was not significantly modified by nandrolone decanoate treatment. Nandrolone decanoate treatment thus affects Ca (2+) uptake by the SR in a fibre-type dependent manner.

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

Science.gov (United States)

Delmotte, Philippe; Sieck, Gary C

2015-02-01

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

International Nuclear Information System (INIS)

de Ancos, J.G.; Inesi, G.

1988-01-01

Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32 P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [ 14 C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis without significant incorporation of the 14 C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [ 14 C]DCCD under more drastic conditions produced inhibition of calcium binding, 14 C radioactive labeling of tryptic fragments A 1 and A 2 (but not of B), and extensive cross-linking. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A 2 , and internal cross-linking of fragment A 1 . It is proposed that both A 1 and A 2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A 2 are directly involved in calcium complexation. A diagram is constructed, representing the relative positions of labels and functional domains within the ATPase protein

Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

Directory of Open Access Journals (Sweden)

Yael Yaniv

Full Text Available Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP.To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84.A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase decreases atrioventricular node-paced heart rate in rabbits.

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Cheng, Hongwei; Smith, Godfrey L; Orchard, Clive H; Hancox, Jules C; Burton, Francis L

2012-10-01

Recent data indicate that Ca(2+) cycling in isolated atrioventricular node (AVN) cells contributes to setting spontaneous rate. The aim of the present study was to extend this observation to the intact AVN in situ, by evaluating the effects of inhibiting sarcoplasmic reticulum Ca(2+) uptake with cyclopiazonic acid (CPA) on intact AVN spontaneous activity and its response to isoprenaline. A model of the AVN-paced heart was produced to investigate intact AVN automaticity, by surgical ablation of the sino-atrial node (SAN) in the rabbit Langendorff-perfused heart. Electrograms were recorded from a site close to the AVN (triangle of Koch), an atrial site above the AVN, the left atrium and right ventricle, enabling AVN pacing of the preparation to be confirmed. Before SAN ablation, the heart rate was 166.8 ± 5.4 beats min(-1). Ablation of the SAN was clearly indicated by a sudden and significant decrease of heart rate to 108.6 ± 9.6 beats min(-1) (P AVN rate to 187.8 ± 12.0 beats min(-1) after 1 min of application (P AVN rate to 81.6 ± 4.8 (n = 9) and 77.4 ± 6.0 beats min(-1) (n = 7), respectively [P AVN rate increase in response to isoprenaline from 78.8 ± 10.0 to 46.8 ± 6.8 and 26.7 ± 5.3%, respectively (P AVN rate and its response to isoprenaline indicate that Ca(2+) cycling is important to the intact AVN spontaneous activity and its acceleration during sympathetic stimulation.

Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

DEFF Research Database (Denmark)

Godiksen, Helene; Jessen, Flemming

2002-01-01

Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes......+- ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degreesC in salmon and rainbow trout, respectively. Ca2+-dependence in the four different fish species investigated was very similar with half maximal activation (K-0.5) between 0.2 and 0.6 muM and half maximal inhibition (I-0.......5) between 60 and 250 muM. Results indicated that interaction between SR Ca2+-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated. (C) 2002 Elsevier Science Inc. All rights reserved....

pH-modulation of chloride channels from the sarcoplasmic reticulum of skeletal muscle.

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Kourie, J I

1999-01-01

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pHcis) and luminal pH (pHtrans) was investigated using the lipid bilayer-vesicle fusion technique. Low pHcis 6.75-4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pHcis 7.26-7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65-75 pS) whereas at low pHcis 6.75-4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5-40 pS). Similarly, low pHtrans 4.07, but not pHtrans 6.28, modified the activity of SCl channels. The effects of low pHcis are pronounced at 10(-3) and 10(-4) M [Ca2+]cis but are not apparent at 10(-5) M [Ca2+]cis, where the subconductances of the channel are already prominent. Low pHcis-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pHcis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

International Nuclear Information System (INIS)

Teruel, J.A.; Inesi, G.

1988-01-01

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca 2+ -ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca 2+ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover

Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography

International Nuclear Information System (INIS)

Andersen, J.P.; Vilsen, B.; Nielsen, H.; Moller, J.V.

1986-01-01

Sarcoplasmic reticulum Ca 2+ -ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca 2+ -ATPase occurred within a few hours in the presence of ≤ 50 μM Ca 2+ . The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high 45 Ca 2+ concentration (500 μM), monomeric Ca 2+ -ATPase was stable for several house. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca 2+ -ATPase was found to be 10 5 -10 6 M -1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca 2+ -ATPase, even above the critical micellar concentration of the detergent. Binding of Ca 2+ and 48 V vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca 2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. The results suggest that formation of Ca 2+ -ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

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Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

2015-12-15

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

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Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J.; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T.; Skurvydas, Albertas; Westerblad, Håkan

2015-01-01

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca2+ leak at rest, and depressed force production due to impaired SR Ca2+ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca2+-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group. PMID:26575622

Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis

International Nuclear Information System (INIS)

McGrew, S.G.; Inui, Makoto; Chadwick, C.C.; Boucek, R.J. Jr.; Jung, C.Y.; Fleischer, S.

1989-01-01

The calcium release channel of sarcoplasmic reticulum which triggers muscle contraction in excitation-contraction coupling has recently been isolated. The channel has been found to be morphologically identical with the feet structures of the junctional face membrane of terminal cisternae and consists of an oligomer of a unique high molecular weight polypeptide. In this study, the authors compare the target size of the calcium release channel from heart and skeletal muscle using target inactivation analysis. The target molecular weights of the calcium release channel estimated by measuring ryanodine binding after irradiation are similar for heart (139,000) and skeletal muscle (143,000) and are smaller than the monomeric unit (estimated to be about 360,000). The target size, estimated by measuring polypeptide remaining after irradiation, was essentially the same for heart and skeletal muscle, 1,061,000 and 1,070,000, respectively, indicating an oligomeric association of protomers. Thus, the calcium release channel of both cardiac and skeletal muscle reacts uniquely with regard to target inactivation analysis in that (1) the size by ryanodine binding is smaller than the monomeric unit and (2) a single hit leads to destruction of more than one polypeptide, by measuring polypeptide remaining. The target inactivation analysis studies indicate that heart and skeletal muscle receptors are structurally very similar

Altered calcium pump and secondary deficiency of γ-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from δ-sarcoglycan knockout mice

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Solares-Pérez, Alhondra; Álvarez, Rocío; Crosbie, Rachelle H.; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J.; Ortega, Alicia; Coral-Vazquez, Ramón

2016-01-01

Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and δ-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of δ-SG isoforms in TT and SR results in a secondary deficiency of γ-SG and µSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the δ-SG isoforms may stabilize the expression of γ-SG and µSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. PMID:20638123

Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: role of nitroso-redox balance.

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Dulce, Raul A; Mayo, Vera; Rangel, Erika B; Balkan, Wayne; Hare, Joshua M

2015-01-02

Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak. We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-). Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR

Effect of triorganotin compounds on calcium transport mechanisms in rat cardiac sarcoplasmic reticulum

International Nuclear Information System (INIS)

Cameron, J.A.; Kodavanti, P.R.S.; Yallapragada, P.R.; Desaiah, D.

1990-01-01

Although organotin compounds, in general, are neurotoxicants, recent studies indicate that these tin compounds affect heme metabolism as well as cardiovascular system. Sarcoplasmic reticulum (SR) calcium pump together with phosphorylation of phospholamban has an important role in myocardial contraction and relaxation. Since organotin compounds interfere with cardiovascular system, we have studied the in vitro as well as in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on cardiac SR Ca 2+ -pump activity, in order to know the relative potency of these tin compounds. SR was isolated from heart ventricles of male Sprague-Dawley rats and used for in vitro studies. For in vivo studies, rats were treated orally in corn oil for 6 days with different doses of TET (0.5, 1.0 and 1.5 mg/kg/d), TMT (0.75, 1.5 and 2.5 mg/kg/d) and TBT (0.75, 1.5 and 2.5 mg/kg/d). Rats were sacrificed 24 hr after the last dosage and cardiac SR was prepared. Cardiac SR Ca 2+ -ATPase and 45 Ca-uptake were measured. All the three tin compounds inhibited Ca 2+ -ATPase and 45 Ca-uptake in vitro in a concentration dependent manner. The order of potency for Ca 2+ -ATPase as determined IC 50 , is TBT (2 uM) > TET (63 uM) > TMT (280 uM). For 45 Ca-uptake, if followed the same order i.e., TBT (0.35 uM) > TET (10 uM) > TMT (440 uM). In agreement with in vitro results, both SR Ca 2+ -ATPase and 45 Ca-uptake were significantly inhibited in rats treated with these tin compounds. These studies indicate that triorganotin compounds affect Ca 2+ -pumping mechanisms and thereby alter cardiac contraction-relaxation process

N-acetylcysteine fails to modulate the in vitro function of sarcoplasmic reticulum of diaphragm in the final phase of fatigue.

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Mishima, T; Yamada, T; Matsunaga, S; Wada, M

2005-07-01

In the present study, we tested the hypothesis whether N-acetylcysteine (NAC), a non-specific antioxidant, might influence fatigue by modulating Ca2+-handling capacity by the sarcoplasmic reticulum (SR). In the presence (10 mm) or absence of NAC, bundles of rat diaphragm were stimulated with tetanic trains (350 ms, 30-40 Hz) at 1 train every 2 s for 300 s. SR functions, as assessed by SR Ca2+-uptake and release rates and SR Ca2+-ATPase activity, were measured in vitro on muscle homogenates. Following the 300-s stimulation, the force developed by NAC-treated muscles is approximately 1.8-fold higher (P depression in SR function (P < 0.05). Despite the differing degrees of fatigue between NAC-treated and non-treated muscles, SR functions in these muscles were reduced to similar extents. These results suggest that modulation of SR function measured in vitro may not be a major contributor to inhibition of diaphragmic fatigue with antioxidant, at least, in the final phase of fatigue where force output is remarkably reduced.

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum

International Nuclear Information System (INIS)

Kasinathan, C.; Kirchberger, M.A.

1988-01-01

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K + ,-Ca 2+ )-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 μmol x mg -1 x h -1 in SR and plasma membrane preparations, respectively, and (Na + , K + )-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 μmol x mg -1 x h -1 , respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0. Ca 2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 μM Ca 2+ . No loss of [ 3 H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca 2+ . The Ca 2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca 2+ ] and became uncompetitive at higher [Ca 2+ ]. It is concluded that CDPdiglyceride-inositol transferase is present on SR membranes and is sensitive to micromolar Ca 2+ . The data are consistent with a putative role for the inhibition of the SR transferase by Ca 2+ and acidic pH in the protection of the SR against calcium overload in ischemic myocardium

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle.

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Manno, Carlo; Sztretye, Monika; Figueroa, Lourdes; Allen, Paul D; Ríos, Eduardo

2013-01-15

The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca(2+)], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca(2+)] were measured integrating Ca(2+) release flux, determined with a cytosolic [Ca(2+)] monitor. Free [Ca(2+)](SR) was measured using the cameleon D4cpv-Casq1. In 34 wild-type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse - when flux was greatest - than at its end, and greater in the ON than in the OFF. In 29 Casq1-null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca(2+)](SR) was tracked with the fast dye fluo-5N. The calcium buffering power, binding capacity and non-linear binding properties of the SR measured here could be accounted for by calsequestrin at the concentration present in mammalian muscle, provided that its properties were substantially different from those found in solution. Its affinity should be higher, or K(D) lower than the conventionally accepted 1 mm; its cooperativity (n in a Hill fit) should be higher and the stoichiometry of binding should be at the higher end of the values derived in solution. The reduction in B during release might reflect changes in calsequestrin conformation upon calcium loss.

Altered sarco(endo)plasmic reticulum calcium adenosine triphosphatase 2a content: Targets for heart failure therapy.

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Liu, Gang; Li, Si Qi; Hu, Ping Ping; Tong, Xiao Yong

2018-05-01

Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is responsible for transporting cytosolic calcium into the sarcoplasmic reticulum and endoplasmic reticulum to maintain calcium homeostasis. Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is the dominant isoform expressed in cardiac tissue, which is regulated by endogenous protein inhibitors, post-translational modifications, hormones as well as microRNAs. Dysfunction of sarco(endo)plasmic reticulum calcium adenosine triphosphatase is associated with heart failure, which makes sarco(endo)plasmic reticulum calcium adenosine triphosphatase a promising target for heart failure therapy. This review summarizes current approaches to ameliorate sarco(endo)plasmic reticulum calcium adenosine triphosphatase function and focuses on phospholamban, an endogenous inhibitor of sarco(endo)plasmic reticulum calcium adenosine triphosphatase, pharmacological tools and gene therapies.

The role of the sarcoplasmic reticulum in the generation of high heart rates and blood pressures in reptiles.

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Galli, Gina L J; Gesser, Hans; Taylor, Edwin W; Shiels, Holly A; Wang, Tobias

2006-05-01

The functional significance of the sarcoplasmic reticulum (SR) in the generation of high heart rates and blood pressures was investigated in four species of reptile; the turtle, Trachemys scripta; the python, Python regius, the tegu lizard, Tupinanvis merianae, and the varanid lizard, Varanus exanthematicus. Force-frequency trials and imposed pauses were performed on ventricular and atrial tissue from each species with and without the SR inhibitor ryanodine, and in the absence and presence of adrenaline. In all species, an imposed pause of 1 or 5 min caused a post-rest decay of force, and a negative force-frequency response was observed in all species within their in vivo frequency range of heart rates. These relationships were not affected by either ryanodine or adrenaline. In ventricular strips from varanid lizards and pythons, ryanodine caused significant reductions in twitch force within their physiologically relevant frequency range. In atrial tissue from the tegu and varanid lizards, SR inhibition reduced twitch force across the whole of their physiological frequency range. In contrast, in the more sedentary species, the turtle and the python, SR inhibition only decreased twitch force at stimulation frequencies above maximal in vivo heart rates. Adrenaline caused an increase in twitch force in all species studied. In ventricular tissue, this positive inotropic effect was sufficient to overcome the negative effects of ryanodine. In atrial tissue however, adrenaline could only ameliorate the negative effects of ryanodine at the lower pacing frequencies. Our results indicate that reptiles recruit Ca2+ from the SR for force development in a frequency and tissue dependent manner. This is discussed in the context of the development of high reptilian heart rates and blood pressures.

Influence of the sarcoplasmic reticulum on the inotropic responses of the rat myocardium resulting from changes in rate and rhythm.

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Mill, J G; Vassallo, D V; Leite, C M; Campagnaro, P

1994-06-01

1. The role of the sarcoplasmic reticulum (SR) in the inotropic responses produced by changes in stimulation rate and rhythm and resting tension was investigated in the rat myocardium. 2. Rat papillary muscles contracting isometrically (basic stimulation rate = 30/min) were superfused in vitro with normal Krebs solution and after addition of ryanodine (1 microM). Post-rest potentiation was obtained after pauses of 5, 10, 15, 30, 60 and 120 s, and the stimulation rate was changed from 6 to 90 bpm. Post-extrasystolic potentiation was induced by interpolating an extra stimulus after an interval of 413 +/- 15 ms. NiCl2 (2 mM) was used to confirm that contractions obtained after SR blockade with ryanodine were activated only by sarcolemmal calcium influx. 3. In the presence of ryanodine, the post-rest potentiation phenomenon disappears and the force-frequency relationship changes from the typical force decrease produced by rate increase to force increase. Under the effect of ryanodine, resting tension increased with the increase in stimulation rate. This behavior was enhanced by reducing extracellular KCl from 5.4 mM to 1 mM. This maneuver decreases Na(+)-K(+)-ATPase and increases intracellular Na+ activity, which reduces Ca2+ extrusion through the Na(+)-Ca2+ exchange mechanism. 4. SR participation in the post-extrasystolic potentiation phenomenon is also suggested because ryanodine treatment reversed the extrasystolic force depression into potentiation. In the presence of ryanodine, blockade of Ca2+ influx with NiCl2 (2 mM) abolished isometric contractions indicating that after SR blockade contractions are mainly dependent on sarcolemmal Ca2+ influx. 5. The results suggest that the SR is involved in the genesis of post-rest potentiation and contributes to the typical force-frequency relationship of the rat myocardium and to the post-extrasystolic potentiation phenomenon. Moreover, SR activity seems to be important for the maintenance of low resting tension in the

Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A2

International Nuclear Information System (INIS)

Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

1986-01-01

Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1- 14 C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A 2 (PLA 2 ) activity (64.3-545.6 nmols/min/mg). The PLA 2 was maximally active in the neutral-alkaline pH range, was Ca 2+ -dependent, and was unaffected by the addition of xanthine. PLA 2 activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA 2 activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca 2+ -dependent PLA 2 measured in various tissue homogenates (≤ 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA 2 may have influenced previously published reports, and such studies should be interpreted cautiously

Decrease in sarcoplasmic reticulum calcium content, not myofilament function, contributes to muscle twitch force decline in isolated cardiac trabeculae

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Milani-Nejad, Nima; Brunello, Lucia; Gyorke, Sándor; Janssen, Paul M.L.

2014-01-01

We set out to determine the factors responsible for twitch force decline in isolated intact rat cardiac trabeculae. The contractile force of trabeculae declined over extended periods of isometric twitch contractions. The force-frequency relationship within the frequency range of 4–8 Hz, at 37 °C, became more positive and the frequency optimum shifted to higher rates with this decline in baseline twitch tensions. The post-rest potentiation (37 °C), a phenomenon highly dependent on calcium handling mechanisms, became more pronounced with decrease in twitch tensions. We show that the main abnormality during muscle run-down was not due to a deficit in the myofilaments; maximal tension achieved using a K+ contracture protocol was either unaffected or only slightly decreased. Conversely, the sarcoplasmic reticulum (SR) calcium content, as assessed by rapid cooling contractures (from 27 °C to 0 °C), decreased, and had a close association with the declining twitch tensions (R2 ~ 0.76). SR Ca2+-ATPase, relative to Na+/Ca2+ exchanger activity, was not altered as there was no significant change in paired rapid cooling contracture ratios. Furthermore, confocal microscopy detected no abnormalities in the overall structure of the cardiomyocytes and t-tubules in the cardiac trabeculae (~23 °C). Overall, the data indicates that the primary mechanism responsible for force run-down in multi-cellular cardiac preparations is a decline in the SR calcium content and not the maximal tension generation capability of the myofilaments. PMID:25056841

Matching of sarcoplasmic reticulum and contractile properties in rat fast- and slow-twitch muscle fibres.

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Trinh, Huong H; Lamb, Graham D

2006-07-01

1. The twitch characteristics (fast-twitch or slow-twitch) of skeletal muscle fibres are determined not only by the contractile apparatus properties of the fibre, but also by the time-course of Ca2+ release and re-uptake by the sarcoplasmic reticulum (SR). The present study examined, in individual fibres from non-transforming muscle of the rat, whether particular SR properties are matched to the contractile apparatus properties of the fibre, in particular in the case of fibres with fast-twitch contractile apparatus located in a slow-twitch muscle, namely the soleus. 2. Force was recorded in single, mechanically skinned fibres from extensor digitorum longus (EDL), gastrocnemius, peroneus longus and soleus muscles. Using repeated cycles in which the SR was emptied of all releasable Ca2+ and then reloaded, it was possible to determine the relative amount of Ca2+ present in the SR endogenously, the maximum SR capacity and the rate of Ca2+ loading. The sensitivity of the contractile apparatus to Ca2+ and Sr2+ was used to classify the fibres as fast-twitch (FT), slow-twitch (ST) or mixed (fibres examined) and thereby identify the likely troponin C and myosin heavy chain types present. 3. There was no significant difference in SR properties between the groups of FT fibres obtained from the four different muscles, including soleus. Despite some overlap in the SR properties of individual fibres between the FT and ST groups, the properties of the FT fibres in all four muscles studied were significantly different from those of the ST and mixed fibres. 4. In general, in FT fibres the SR had a larger capacity and the endogenous Ca2+ content was a relatively lower percentage of maximum compared with ST fibres. Importantly, in terms of their SR properties, FT fibres from soleus muscle more closely resembled FT fibres from other muscles than they did ST fibres from soleus muscle.

Effects of dantrolene and its derivatives on Ca2+ release from the sarcoplasmic reticulum of mouse skeletal muscle fibres

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Ikemoto, Takaaki; Hosoya, Takamitsu; Aoyama, Hiroshi; Kihara, Yasutaka; Suzuki, Masaaki; Endo, Makoto

2001-01-01

We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca2+ release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle.In intact muscles, GIF-0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF-0082, an azido-functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF-0185.In skinned fibres, Dan inhibited Ca2+-induced Ca2+ release (CICR) under Mg2+-free conditions at room temperature. In contrast, GIF-0082 and GIF-0185 showed no inhibitory effect on CICR under the same conditions.Dan-induced inhibition of CICR was not affected by the presence of GIF-0082, whereas it was diminished in the presence of GIF-0185.GIF-0082 and GIF-0185 significantly inhibited clofibric acid (Clof)-induced Ca2+ release, as did Dan.Several Dan derivatives other than GIF-0082 and GIF-0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof-induced Ca2+ release.The magnitudes of inhibition of Clof-induced Ca2+ release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof-induced opening of the RyR-Ca2+ release channel may be similar to that of physiological Ca2+ release (PCR).These results indicate that the difference in opening modes of the RyR-Ca2+ release channel is recognized by certain Dan derivatives. PMID:11606312

Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle.

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Baylor, S M; Hollingworth, S

2003-08-15

Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 degrees C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Delta[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 +/- 1.0 microM (mean +/- S.E.M.); half-duration, 7.7 +/- 0.6 ms; fast-twitch: peak amplitude 18.5 +/- 0.5 microM; half-duration, 4.9 +/- 0.3 ms). SR Ca2+ release was estimated from Delta[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Delta[CaT]) and the peak rate of Ca2+ release ((d/dt)Delta[CaT]) differed about 3-fold between the fibre types (slow-twitch: Delta[CaT], 127 +/- 7 microM; (d/dt)Delta[CaT], 70 +/- 6 microM ms-1; fast-twitch: Delta[CaT], 346 +/- 6 microM; (d/dt)Delta[CaT], 212 +/- 4 microM ms-1). In contrast, the half-duration of (d/dt)Delta[CaT] was very similar in the two fibre types (slow-twitch, 1.8 +/- 0.1 ms; fast-twitch, 1.6 +/- 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Delta[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2-0.3; fast-twitch, 0.1-0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.

A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

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Tobias Gröβl

Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

Properties of Ca2+ release induced by clofibric acid from the sarcoplasmic reticulum of mouse skeletal muscle fibres

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Ikemoto, Takaaki; Endo, Makoto

2001-01-01

To characterize the effect of clofibric acid (Clof) on the Ca2+ release mechanism in the sarcoplasmic reticulum (SR) of skeletal muscle, we analysed the properties of Clof-induced Ca2+ release under various conditions using chemically skinned skeletal muscle fibres of the mouse.Clof (>0.5 mM) released Ca2+ from the SR under Ca2+-free conditions buffered with 10 mM EGTA (pCa >8).Co-application of ryanodine and Clof at pCa >8 but not ryanodine alone reduced the Ca2+ uptake capacity of the SR. Thus, Ca2+ release induced by Clof at pCa >8 must be a result of the activation of the ryanodine receptor (RyR).At pCa >8, (i) Clof-induced Ca2+ release was inhibited by adenosine monophosphate (AMP), (ii) the inhibitory effect of Mg2+ on the Clof-induced Ca2+ release was saturated at about 1 mM, and (iii) Clof-induced Ca2+ release was not inhibited by procaine (10 mM). These results indicate that Clof may activate the RyR-Ca2+ release channels in a manner different from Ca2+-induced Ca2+ release (CICR).In addition to this unique mode of opening, Clof also enhanced the CICR mode of opening of RyR-Ca2+ release channels.Apart from CICR, a high concentration of Ca2+ might also enhance the unique mode of opening by Clof.These results suggest that some features of Ca2+ release activated by Clof are similar to those of physiological Ca2+ release (PCR) in living muscle cells and raise the possibility that Clof may be useful in elucidating the mechanism of PCR in skeletal muscle. PMID:11606311

Role of sarcoplasmic reticulum calcium in development of secondary calcium rise and early afterdepolarizations in long QT syndrome rabbit model.

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Po-Cheng Chang

Full Text Available L-type calcium current reactivation plays an important role in development of early afterdepolarizations (EADs and torsades de pointes (TdP. Secondary intracellular calcium (Cai rise is associated with initiation of EADs.To test whether inhibition of sarcoplasmic reticulum (SR Ca2+ cycling suppresses secondary Cai rise and genesis of EADs.Langendorff perfusion and dual voltage and Cai optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB was created by radiofrequency ablation. After baseline studies, E4031, SR Ca2+ cycling inhibitors (ryanodine plus thapsigargin and nifedipine were then administrated subsequently, and the protocols were repeated.At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD was significantly prolonged and the amplitude of secondary Cai rise was enhanced, and 7 (70% rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced (2 hearts, 20%, p = 0.03. Although APD was significantly prolonged (from 298 ± 30 ms to 457 ± 75 ms at pacing cycle length of 1000 m, p = 0.007 by ryanodine plus thapsigargin, the secondary Cai rise was suppressed (from 8.8 ± 2.6% to 1.2 ± 0.9%, p = 0.02. Nifedipine inhibited TdP inducibility in all rabbit hearts.In this AVB and long QT rabbit model, inhibition of SR Ca2+ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Cai rise and genesis of EADs.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

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Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

2012-05-01

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of the separate lipid and protein profile structures derived from the total membrane profile structure or isolated sarcoplasmic reticulum via x-ray and neutron diffraction

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Herbette, L.; Blasie, J.K.

1984-01-01

Sarcoplasmic reticulum (SR) membranes were prepared to contain biosynthetically deuterated SR phospholipids utilizing specific and general phospholipid exchange proteins (PLEP). Functional measurements and freeze fracture on SR dispersions and x-ray diffraction of hydrated oriented membrane multilayers revealed that the exchanged SR membranes were very similar to unexchanged SR membranes. Low resolution (28-A) neutron diffraction studies utilizing SR membranes exchanged with either protonated or perdeuterated SR phospholipids allowed direct determination of the lipid profile within the isolated SR membrane at two different unit cell repeat distances. These lipid profile structures were found to be highly asymmetric regarding the conformation of the fatty acid chain extents and compositional distribution of phospholipid molecules in the inner vs. outer monolayer of the SR membrane bilayer. The relatively high resolution (11-A) electron-density profile from x-ray diffraction was decomposed by utilizing the asymmetry in the number of phospholipid molecules residing in the inner vs. outer monolayer of the SR lipid bilayer as obtained from the neutron diffraction study. To our knowledge, this represents the first direct determination of a lipid bilayer profile structure within an isolated membrane system

Impact of sarcoplasmic reticulum calcium release on calcium dynamics and action potential morphology in human atrial myocytes: a computational study.

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Jussi T Koivumäki

Full Text Available Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca²+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL ion currents, accounts for the heterogeneity of intracellular Ca²+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR. Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca²+ dynamics: 1 the biphasic increment during the upstroke of the Ca²+ transient resulting from the delay between the peripheral and central SR Ca²+ release, and 2 the relative contribution of SL Ca²+ current and SR Ca²+ release to the Ca²+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca²+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca²+ release sites define the interface between Ca²+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca²+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca²+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca²+ signaling. Thus, the model provides a useful framework for future

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat.

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Murphy, Robyn M; Larkins, Noni T; Mollica, Janelle P; Beard, Nicole A; Lamb, Graham D

2009-01-15

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 +/- 2 and 10 +/- 1 micromol (l fibre volume)(-1), quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (approximately 20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca2+-ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (approximately 20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1

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Glukhov, Alexey V.; Kalyanasundaram, Anuradha; Lou, Qing; Hage, Lori T.; Hansen, Brian J.; Belevych, Andriy E.; Mohler, Peter J.; Knollmann, Björn C.; Periasamy, Muthu; Györke, Sandor; Fedorov, Vadim V.

2015-01-01

Aims Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. Methods and results In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca2+ cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2−/−) mice. Casq2−/− mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca2+ cycling, including abnormal Ca2+ release, periods of significantly elevated diastolic Ca2+ levels leading to pauses and unstable pacemaker rate. Importantly, Ca2+ cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca2+ transient upstrokes throughout the atrial pacemaker complex. Conclusions Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca2+ release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients. PMID:24216388

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase.

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Vilsen, B; Andersen, J P

1987-12-30

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of

A low-dose β1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

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Shigeki Kobayashi

Full Text Available OBJECTIVES: The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. BACKGROUND: The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca(2+ handling in heart failure remains unclear. METHODS: We investigated the effect of milrinone plus landiolol on intracellular Ca(2+ transient (CaT, cell shortening (CS, the frequency of diastolic Ca(2+ sparks (CaSF, and sarcoplasmic reticulum Ca(2+ concentration ({Ca(2+}SR in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2 and phospholamban (PLB. RESULTS: In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808 in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17. Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz in failing cardiomyocytes. CONCLUSION: A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+ leak.

A low-dose β1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

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Kobayashi, Shigeki; Susa, Takehisa; Ishiguchi, Hironori; Myoren, Takeki; Murakami, Wakako; Kato, Takayoshi; Fukuda, Masakazu; Hino, Akihiro; Suetomi, Takeshi; Ono, Makoto; Uchinoumi, Hitoshi; Tateishi, Hiroki; Mochizuki, Mamoru; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Yamamoto, Takeshi; Yano, Masafumi

2015-01-01

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca(2+) handling in heart failure remains unclear. We investigated the effect of milrinone plus landiolol on intracellular Ca(2+) transient (CaT), cell shortening (CS), the frequency of diastolic Ca(2+) sparks (CaSF), and sarcoplasmic reticulum Ca(2+) concentration ({Ca(2+)}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB). In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+)}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+)}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes. A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+) leak.

Time Course of the Response of Myofibrillar and Sarcoplasmic Protein Metabolism to Unweighting of the Soleus Muscle

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Munoz, Kathryn A.; Satarug, Soisungwan; Tischler, Marc E.

1993-01-01

Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of H-3-phenylaianine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

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Roewer Norbert

2005-06-01

Full Text Available Abstract Background Malignant hyperthermia (MH is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible, 7 MHE (equivocal and 16 MHN (non-susceptible classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM and halothane (0.11; 0.22; 0.44; 0.66 mM was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p Results Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.

Cannabinoid signalling inhibits sarcoplasmic Ca2+ release and regulates excitation–contraction coupling in mammalian skeletal muscle

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Oláh, Tamás; Bodnár, Dóra; Tóth, Adrienn; Vincze, János; Fodor, János; Reischl, Barbara; Kovács, Adrienn; Ruzsnavszky, Olga; Dienes, Beatrix; Szentesi, Péter; Friedrich, Oliver

2016-01-01

Key points Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation–contraction coupling (ECC) of mammalian skeletal muscle remains unknown.We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+‐sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue.We demonstrate that CB1Rs are not connected to the inositol 1,4,5‐trisphosphate pathway either in myotubes or in adult muscle fibres.By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein‐mediated way in adult skeletal muscle fibres but not in myotubes.These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs. Abstract Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R‐mediated signalling affects the contraction and Ca²⁺ homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+‐sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ transients, nor did they alter excitation–contraction coupling. By contrast, in isolated muscle fibres of wild‐type mice, although CB1R agonists did not evoke IP3

Cannabinoid signalling inhibits sarcoplasmic Ca2+ release and regulates excitation-contraction coupling in mammalian skeletal muscle.

Science.gov (United States)

Oláh, Tamás; Bodnár, Dóra; Tóth, Adrienn; Vincze, János; Fodor, János; Reischl, Barbara; Kovács, Adrienn; Ruzsnavszky, Olga; Dienes, Beatrix; Szentesi, Péter; Friedrich, Oliver; Csernoch, László

2016-12-15

Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation-contraction coupling (ECC) of mammalian skeletal muscle remains unknown. We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca 2+ -sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue. We demonstrate that CB1Rs are not connected to the inositol 1,4,5-trisphosphate pathway either in myotubes or in adult muscle fibres. By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca 2+ release and sarcoplasmic reticulum Ca 2+ ATPase during ECC in a G i/o protein-mediated way in adult skeletal muscle fibres but not in myotubes. These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs. Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R-mediated signalling affects the contraction and Ca²⁺ homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca 2+ -sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5-trisphosphate (IP 3 )-mediated Ca 2+ transients, nor did they alter excitation-contraction coupling. By contrast, in isolated muscle fibres of wild-type mice, although CB1R agonists did not evoke IP 3 -mediated Ca 2

Contractile properties and sarcoplasmic reticulum calcium content in type I and type II skeletal muscle fibres in active aged humans.

Science.gov (United States)

Lamboley, C R; Wyckelsma, V L; Dutka, T L; McKenna, M J; Murphy, R M; Lamb, G D

2015-06-01

Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres. This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults. The maximum level of force production (per unit cross-sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium. The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast- and slow-twitch muscle fibres in the Old subjects. These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres. This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca(2+) content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ∼17% and Ca(2+) sensitivity was also reduced (pCa50 decreased ∼0.05 pCa units) relative to that in Young. S-Glutathionylation of fast troponin I (TnIf ) markedly increased Ca(2+) sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca(2+) content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca(2+) by a combined caffeine and low Mg(2+) stimulus, whereas in fibres of Old the amount of non-releasable Ca(2+) was significantly increased (by > 12% of endogenous Ca(2+) content). Western

The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm

DEFF Research Database (Denmark)

Winther, Anne-Marie Lund; Bublitz, Maike; Karlsen, Jesper Lykkegaard

2013-01-01

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal...

Muscle Glycogen Content Modifies SR Ca2 + Release Rate in Elite Endurance Athletes

DEFF Research Database (Denmark)

Gejl, Kasper Degn; Hvid, Lars G; Frandsen, Ulrik

2014-01-01

The aim of the present study was to investigate the influence of muscle glycogen content on sarcoplasmic reticulum (SR) function and peak power output (Wpeak) in elite endurance athletes.......The aim of the present study was to investigate the influence of muscle glycogen content on sarcoplasmic reticulum (SR) function and peak power output (Wpeak) in elite endurance athletes....

Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability.

Science.gov (United States)

Li, Meng; Li, Zheng; Li, Xin; Xin, Jianzeng; Wang, Ying; Li, Guixia; Wu, Liguo; Shen, Qingwu W; Zhang, Dequan

2018-02-01

The phosphorylation of sarcoplasmic proteins in postmortem muscles was investigated in relationship to color stability in the present study. Although no difference was observed in the global phosphorylation level of sarcoplasmic proteins, difference was determined in the phosphorylation levels of individual protein bands from muscles with different color stability. Correlation analysis and liquid chromatography - tandem mass spectrometry (LC-MS/MS) identification of phosphoproteins showed that most of the color stability-related proteins were glycolytic enzymes. Interestingly, the phosphorylation level of myoglobin was inversely related to meat color stability. As the phosphorylation of myoglobin increased, color stability based on a ∗ value decreased and metMb content increased. In summary, the study revealed that protein phosphorylation might play a role in the regulation of meat color stability probably by regulating glycolysis and the redox stability of myoglobin, which might be affected by the phosphorylation of myoglobin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Solution-blown nanofiber mats from fish sarcoplasmic protein

DEFF Research Database (Denmark)

Sett, S.; Boutrup Stephansen, Karen; Yarin, A.L.

2016-01-01

In the present work, solution-blowing was adopted to form nanofibers from fish sarcoplasmic proteins (FSPs). Nanofiber mats containing different weight ratios (up to 90/10) of FSP in the FSP/nylon 6 blended nanofibers were formed from formic acid solutions, and compared to electrospun fibers made...... that the production rate of solution-blowing was increased 30-fold in relation to electrospinning. Overall, this study reveals FSP as an interesting biopolymeric alternative to synthetic polymers, and the introduction of FSP to nylon 6 provides a composite with controlled properties....

Temperature dependence of the kinetics of isometric myocardium relaxation

Energy Technology Data Exchange (ETDEWEB)

Izakov, V.Ya.; Bykov, B.L.; Kimmelman, I.Ya.

1981-11-01

The dependence of the exponential decay constant expressing the isometric relaxation of the myocardium on temperature is investigated in animals with various specific contents of myocardial sarcoplasmic reticulum. Experiments were performed on cardiac ventricles and atria isolated from rabbits, frogs and turtles and electrically stimulated to produce maximal contraction at temperatures from 10 to 35 C. Arrhenius plots derived from the data are found to be linear in the myocardia of the rabbit and frog, with a greater activation energy for the relaxation found in the rabbit. The Arrhenius plot for the turtle, which has a sarcoplasmic reticulum content intermediate between those of the frog and rabbit, corresponds to two straight lines with different activation energies. Results thus support the hypothesis of two separate mechanisms of calcium removal, involving the sarcoplasmic reticulum and cellular membrane, in muscle relaxation.

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.

Science.gov (United States)

Peter, Angela K; Miller, Gaynor; Capote, Joana; DiFranco, Marino; Solares-Pérez, Alhondra; Wang, Emily L; Heighway, Jim; Coral-Vázquez, Ramón M; Vergara, Julio; Crosbie-Watson, Rachelle H

2017-06-06

Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd -/- ) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. Alternative splicing of proteins from the SG

Picture frame fibres in a carrier of the trait for malignant hyperpyrexia

Energy Technology Data Exchange (ETDEWEB)

Isaacs, H; Badenhorst, M [University of the Witwatersrand, Johannesburg (South Africa). Department of Physiology; Heffron, J J.A. [University of the Witwatersrand, Johannesburg (South Africa). Department of Physiological Chemistry

1975-11-01

A member of a family which was known to be susceptible to malignant hyperpyrexia, who was identified as a carrier by the presence of an elevated serum creatinephosphokinase, has been investigated further. Muscle was examined biochemically, and the study included the sarcoplasmic ATPase-activity, actinomycin, Mg2+ ATPase activity, ATP, phosphocreatine and glucose-6-phosphate. In addition, the calcium uptake by the sarcoplasmic reticulum was studied. The histochemical analysis of the muscle revealed the presence of a new fibre type characterized by a dense rim of ATPase activity, which gives the impression of a 'picture-frame'. Ultramicroscopic study revealed changes in the mitochondria and areas of myofibrillar disruption with swelling of the sarcoplasmic reticulum.

Picture frame fibres in a carrier of the trait for malignant hyperpyrexia

International Nuclear Information System (INIS)

Isaacs, H.; Badenhorst, M.; Heffron, J.J.A.

1975-01-01

A member of a family which was known to be susceptible to malignant hyperpyrexia, who was identified as a carrier by the presence of an elevated serum creatinephosphokinase, has been investigated further. Muscle was examined biochemically, and the study included the sarcoplasmic ATPase-activity, actinomycin, Mg2+ ATPase activity, ATP, phosphocreatine and glucose-6-phosphate. In addition, the calcium uptake by the sarcoplasmic reticulum was studied. The histochemical analysis of the muscle revealed the presence of a new fibre type characterized by a dense rim of ATPase activity, which gives the impression of a 'picture-frame'. Ultramicroscopic study revealed changes in the mitochondria and areas of myofibrillar disruption with swelling of the sarcoplasmic reticulum

Is contraction-stimulated glucose transport feedforward regulated by Ca²⁺?

DEFF Research Database (Denmark)

Jensen, Thomas Elbenhardt; Angin, Yeliz; Sylow, Lykke

2014-01-01

cell types. The literature is contrasted against our recent findings suggesting that SR Ca(2+) release is neither essential nor adequate to stimulate glucose transport in muscle. Instead, feedback signals through AMPK and mechanical stress are likely to account for most of contraction......In many cell types, Ca(2+) signals to increase the movement and surface membrane insertion of vesicles. In skeletal muscle, Ca(2+) is predominantly released from the sarcoplasmic reticulum (SR) to initiate contraction. Sarcoplasmic reticulum Ca(2+) release is widely believed to be a direct......-stimulated glucose transport. A revised working model is proposed, in which muscle glucose transport during contraction is not directly regulated by SR Ca(2+) release but rather responds exclusively to feedback signals activated secondary to cross-bridge cycling and tension development....

ALUMINUM CHLORIDE EFFECT ON Ca2+,Mg(2+)-ATPase ACTIVITY AND DYNAMIC PARAMETERS OF SKELETAL MUSCLE CONTRACTION.

Science.gov (United States)

Nozdrenko, D M; Abramchuk, O M; Soroca, V M; Miroshnichenko, N S

2015-01-01

We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10(-4) M Increasing the concentration of AlCl3 to 10(-2) M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg(2+)-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg(2+)-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

Aluminum chloride effect on Ca(2+,Mg(2+-ATPase activity and dynamic parameters of skeletal muscle contraction

Directory of Open Access Journals (Sweden)

D. M. Nozdrenko

2015-10-01

Full Text Available We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10-4 M. Increasing the concentration of AlCl3 to 10-2 M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg2+-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg2+-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

[Changes induced by hypertonic solutions in the transportation of calcium by the cardiac reticular sarcoplasma].

Science.gov (United States)

Sierra, M; Holguín, J A

1979-01-01

In the sarcoplasmic reticulum of the myocardium, celular organell which function is to regulate the cytoplasmic concentration of calcium in contraction and relaxation, we have studied the effect of hypertonic solutions of sucrose between 1 and 6.96 times the normal tonicity in order to observe the behavior of the internal linked or free calcium of this structure, as well as to prove the hypothesis that hypertonic solutions encourage the calcium exit of the sarcoplasmatic reticulum with the resulting signs of contractures. The following results were obtained: 1. The ATP hydrolisis and calcium transport rate are 14% and 90% respectively of the maximum speeds of 10(-5) M in calcium, while for concentrations of 10(-7) M or ess of the said cation, the transport rates and the ATPase do not reach 5% of the maximum values. 2. Between 1 and 2.54 times of the normal tonicity the calcium uptake remains between 400 and 500 nmoles of calcium/mg protein/min, the transported amount of calcium varies between 14 and 16 nmoles/mg protein and the rate of the ATP hydrolysis increases a 37% to 0.4 M in sucrose. 3. Between 0.4 and 1.2 M in sucrose of 2.54 to 6.96 times the isotonicity, the calcium transport rate velocity as well as the ATP hydrolisis are strongly inhibited. The vesicles volume minimizes and the amount of linked calcium remains within the control values, proving that the capacity of linking this cathion is independent from sarcoplasmic reticulum volume. These results show that the sarcoplasmic reticulum is involved in the contractures induced by hypertonic solutions in intact cells, since the osmolarity increase produces changes of volume which results in a decrease of the calcium transportation velocity or in an increase of the exit of said cathion.

A model of propagating calcium-induced calcium release mediated by calcium diffusion

NARCIS (Netherlands)

Backx, P. H.; de Tombe, P. P.; van Deen, J. H.; Mulder, B. J.; ter Keurs, H. E.

1989-01-01

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium

Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation.

NARCIS (Netherlands)

Vattemi, G.; Gualandi, F.; Oosterhof, A.; Marini, M.; Tonin, P.; Rimessi, P.; Neri, M.; Guglielmi, V.; Russignan, A.; Poli, C.; Kuppevelt, A.H.M.S.M. van; Ferlini, A.; Tomelleri, G.

2010-01-01

Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein.

Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

Science.gov (United States)

Pitake, Saumitra; Ochs, Raymond S

2016-04-01

The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation-contraction coupling. However, the mechanism for subsequent Ca(2+) release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca(2+) under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation-contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca(2+) from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca(2+) was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca(2+) concentration in the media. Ca(2+) entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca(2+) entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca(2+) release from the ryanodine receptor to the cytosol. © 2015 by the Society for Experimental Biology and Medicine.

Repeated static contractions increase mitochondrial vulnerability toward oxidative stress in human skeletal muscle

DEFF Research Database (Denmark)

Sahlin, Kent; Nielsen, Jens Steen; Mogensen, Martin

2006-01-01

Repeated static contractions (RSC) induce large fluctuations in tissue oxygen tension and increase the generation of reactive oxygen species (ROS). This study investigated the effect of RSC on muscle contractility, mitochondrial respiratory function, and in vitro sarcoplasmic reticulum (SR) Ca(2......+) kinetics in human muscle. Ten male subjects performed five bouts of static knee extension with 10-min rest in between. Each bout of RSC (target torque 66% of maximal voluntary contraction torque) was maintained to fatigue. Muscle biopsies were taken preexercise and 0.3 and 24 h postexercise from vastus...... lateralis. Mitochondria were isolated and respiratory function measured after incubation with H(2)O(2) (HPX) or control medium (Con). Mitochondrial function was not affected by RSC during Con. However, RSC exacerbated mitochondrial dysfunction during HPX, resulting in decreased respiratory control index...

The role of the endoplasmic reticulum stress response following cerebral ischemia.

Science.gov (United States)

Hadley, Gina; Neuhaus, Ain A; Couch, Yvonne; Beard, Daniel J; Adriaanse, Bryan A; Vekrellis, Kostas; DeLuca, Gabriele C; Papadakis, Michalis; Sutherland, Brad A; Buchan, Alastair M

2018-06-01

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Type 2 diabetes mellitus induces congenital heart defects in murine embryos by increasing oxidative stress, endoplasmic reticulum stress, and apoptosis.

Science.gov (United States)

Wu, Yanqing; Reece, E Albert; Zhong, Jianxiang; Dong, Daoyin; Shen, Wei-Bin; Harman, Christopher R; Yang, Peixin

2016-09-01

Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects remain largely unknown. We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects. A mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination. Maternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin protein; endoplasmic reticulum chaperone gene

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA.

Directory of Open Access Journals (Sweden)

Sen Li

Full Text Available Intracellular pH (pHi and Ca(2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+. The sources of the Ca(2+ increase are from the endoplasmic reticulum (ER Ca(2+ pools as well as from Ca(2+ influx. The store-mobilization component of the Ca(2+ increase induced by the pHi rise was not sensitive to antagonists for either IP(3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA, leading to depletion of the ER Ca(2+ store. We further showed that the physiological consequence of depletion of the ER Ca(2+ store by pHi rise is the activation of store-operated channels (SOCs of Orai1 and Stim1, leading to increased Ca(2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+ leak from ER pools followed by Ca(2+ influx via SOCs.

Insulin-like growth factor stimulation increases radiosensitivity of a pancreatic cancer cell line through endoplasmic reticulum stress under hypoxic conditions

International Nuclear Information System (INIS)

Isohashi, Fumiaki; Endo, Hiroko; Mukai, Mutsuko; Inoue, Masahiro; Inoue, Takehiro

2008-01-01

Tumor hypoxia is an obstacle to radiotherapy. Radiosensitivity under hypoxic conditions is determined by molecular oxygen levels, as well as by various biological cellular responses. The insulin-like growth factor (IGF) signaling pathway is a widely recognized survival signal that confers radioresistance. However, under hypoxic conditions the role of IGF signaling in radiosensitivity is still poorly understood. Here, we demonstrate that IGF-II stimulation decreases clonogenic survival under hypoxic conditions in the pancreatic cancer cell lines AsPC-1 and Panc-1, and in the human breast cancer cell line MCF-7. IGF treatment under hypoxic conditions suppressed increased radiation sensitivity in these cell lines by pharmacologically inhibiting the phosphoinositide 3-kinase-mammalian target of rapamycin pathway, a major IGF signal-transduction pathway. Meanwhile, IGF-II induced the endoplasmic reticulum stress response under hypoxia, including increased protein levels of CHOP and ATF4, mRNA levels of CHOP, GADD34, and BiP as well as splicing levels of XBP-1. The response was suppressed by inhibiting phosphoinositide 3-kinase and mammalian target of rapamycin activity. Overexpression of CHOP in AsPC-1 cells increased radiation sensitivity by IGF-II simulation under hypoxic conditions, whereas suppression of CHOP expression levels with small hairpin RNA or a dominant negative form of a proline-rich extensin-like receptor protein kinase in hypoxia decreased IGF-induced radiosensitivity. IGF-induced endoplasmic reticulum stress contributed to radiosensitization independent of cell cycle status. Taken together, IGF stimulation increased radiosensitivity through the endoplasmic reticulum stress response under hypoxic conditions. (author)

Redox regulation of calcium release in skeletal and cardiac muscle

Directory of Open Access Journals (Sweden)

CECILIA HIDALGO

2002-01-01

Full Text Available In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist and Mg2+ (endogenous inhibitor on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 µM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 µM [Ca2+]. In 10 µM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] ­ 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 µM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed

Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

OpenAIRE

Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

2004-01-01

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine bind...

Progranulin causes adipose insulin resistance via increased autophagy resulting from activated oxidative stress and endoplasmic reticulum stress.

Science.gov (United States)

Guo, Qinyue; Xu, Lin; Li, Huixia; Sun, Hongzhi; Liu, Jiali; Wu, Shufang; Zhou, Bo

2017-01-31

Progranulin (PGRN) has recently emerged as an important regulator for insulin resistance. However, the direct effect of progranulin in adipose insulin resistance associated with the autophagy mechanism is not fully understood. In the present study, progranulin was administered to 3T3-L1 adipocytes and C57BL/6 J mice with/without specific inhibitors of oxidative stress and endoplasmic reticulum stress, and metabolic parameters, oxidative stress, endoplasmic reticulum stress and autophagy markers were assessed. Progranulin treatment increased iNOS expression, NO synthesis and ROS generation, and elevated protein expressions of CHOP, GRP78 and the phosphorylation of PERK, and caused a significant increase in Atg7 and LC3-II protein expression and a decreased p62 expression, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake, demonstrating that progranulin activated oxidative stress and ER stress, elevated autophagy and induced insulin insensitivity in adipocytes and adipose tissue of mice. Interestingly, inhibition of iNOS and ER stress both reversed progranulin-induced stress response and increased autophagy, protecting against insulin resistance in adipocytes. Furthermore, the administration of the ER stress inhibitor 4-phenyl butyric acid reversed the negative effect of progranulin in vivo. Our findings showed the clinical potential of the novel adipokine progranulin in the regulation of insulin resistance, suggesting that progranulin might mediate adipose insulin resistance, at least in part, by inducing autophagy via activated oxidative stress and ER stress.

A novel heterozygous mutation of the WFS1 gene leading to constitutive endoplasmic reticulum stress is the cause of Wolfram syndrome.

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Morikawa, Shuntaro; Tajima, Toshihiro; Nakamura, Akie; Ishizu, Katsura; Ariga, Tadashi

2017-12-01

Wolfram syndrome (WS) is a disorder characterized by the association of insulin-dependent diabetes mellitus (DM), diabetes insipidus, deafness, and optic nerve atrophy. WS is caused by WFS1 mutations encoding WFS1 protein expressed in endoplasmic reticulum (ER). During ER protein synthesis, misfolded and unfolded proteins accumulate, known as "ER stress". This is attenuated by the unfolded protein response (UPR), which recovers and maintains ER functions. Because WFS1 is a UPR component, mutant WFS1 might cause unresolvable ER stress conditions and cell apoptosis, the major causes underlying WS symptoms. We encountered an 11-month-old Japanese female WS patient with insulin-dependent DM, congenital cataract and severe bilateral hearing loss. Analyze the WFS1 and functional consequence of the patient WFS1 in vitro. The patient WFS1 contained a heterozygous 4 amino acid in-frame deletion (p.N325_I328del). Her mutant WFS1 increased GRP78 and ATF6α promoter activities in the absence of thapsigargin, indicating constitutive ER stress and nuclear factor of activated T-cell reporter activity, reflecting elevated cytosolic Ca 2+ signals. Mutant transfection into cells reduced mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca 2+ transport ATPase 2b (SERCA2b) compared with wild type. Because SERCA2b is required for ER and cytoplasmic Ca 2+ homeostasis, decreased SERCA2b expression might affect ER Ca 2+ efflux, causing cell apoptosis. A novel heterozygous mutation of WFS1 induced constitutive ER stress through ATF6α activation and ER Ca 2+ efflux, resulting in cell apoptosis. These results provide new insights into the roles of WFS1 in UPR and mechanism of monogenic DM. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

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Wei XIN

2011-04-01

Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

DEFF Research Database (Denmark)

Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels

2014-01-01

In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator...... of sarcoplasmic reticulum Ca(2+) release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca(2+) ([Ca(2+)]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz...... in tetanic [Ca(2+)]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca(2+)]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca(2+) release critically depends on energy supply from...

Endoplasmic reticulum stress in pathogenesis of diabetic retinopathy and effect of calcium dobesilate

Institute of Scientific and Technical Information of China (English)

Yu-Min Gui; Ming Zhao; Jie Ding

2016-01-01

Objective:To study the mechanism of endoplasmic reticulum stress in the pathogenesis of diabetic retinopathy and effect of calcium dobesilate.Methods:A total of 120 diabetic retinopathy patients treated in our hospital from January 2010 to September 2015 were enrolled in this article. The serum endoplasmic reticulum stress protein and interleukin protein expression levels were analyzed before and after calcium dobesilate treatment. A total of 55 cases of healthy subjects receiving physical examination in our hospital during the same period were taken as control group.Results:Serum endoplasmic reticulum stress proteins PERK, CHOP and IRE as well as interleukin proteins IL1, IL2, IL6 and IL10 expression significantly increased, serum MDA level significantly increased while SOD, CAT and GSHpx levels significantly decreased in diabetic retinopathy patients, and compared with control group (P<0.01); after calcium dobesilate treatment, above factors were significantly restored (P<0.01).Conclusions: Diabetic retinopathy is closely related to endoplasmic reticulum stress and calcium dobesilate treatment may improve diabetic retinopathy by inhibiting endoplasmic reticulum stress.

Inhibition of NAPDH Oxidase 2 (NOX2 Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

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Leroy C Joseph

Full Text Available Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

SR calcium handling and calcium after-transients in a rabbit model of heart failure

NARCIS (Netherlands)

Baartscheer, Antonius; Schumacher, Cees A.; Belterman, Charly N. W.; Coronel, Ruben; Fiolet, Jan W. T.

2003-01-01

Objective: After-depolarization associated arrhythmias are frequently observed in heart failure and associated with spontaneous calcium release from sarcoplasmic reticulum (SR), calcium after-transients. We hypothesize that disturbed SR calcium handling underlies calcium after-transients in heart

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle.

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Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

2004-03-01

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation

NARCIS (Netherlands)

Sood, Subeena; Chelu, Mihail G.; van Oort, Ralph J.; Skapura, Darlene; Santonastasi, Marco; Dobrev, Dobromir; Wehrens, Xander H. T.

2008-01-01

BACKGROUND: Although defective Ca(2+) homeostasis may contribute to arrhythmogenesis in atrial fibrillation (AF), the underlying molecular mechanisms remain poorly understood. Studies in patients with AF revealed that impaired diastolic closure of sarcoplasmic reticulum (SR) Ca(2+)-release channels

Toxicologic and Analytical Studies with T-2 and Related Trichothecene Mycotoxins

Science.gov (United States)

1985-08-20

damaged myocytes, hypercontraction bands with myofibrillar, lysis or marked distension of sarcoplasmic reticulum with myofibrillar ly Is. * ’was...the increased activity of AST in both groups may suggest toxic or ischemic damage ’to visceral or muscular tissues. In the low dose group, systemic...pig. If there is a sudden change In the respiratory requirements, th? air accessory hose (Figure 3) can be used to rapidly add air to the distensible

Genetics Home Reference: Brody myopathy

Science.gov (United States)

... 1 (SERCA1). The SERCA1 enzyme is found in skeletal muscle cells, specifically in the membrane of a structure called the sarcoplasmic reticulum . This structure plays a major role in muscle contraction and relaxation by storing and releasing positively charged ...

Effects of Preslaughter Stress Levels on the Post-mortem Sarcoplasmic Proteomic Profile of Gilthead Seabream Muscle

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Silva, Tomé Santos; Cordeiro, Odete D; Matos, Elisabete D.

2012-01-01

identification was performed by MALDI-TOF-TOF MS. Analysis of the results indicates changes on several cellular pathways, with some of these changes being attributable to oxidative and proteolytic activity on sarcoplasmic proteins, together with leaking of myofibrillar proteins. These processes appear to have...

Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes

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Boutrup Stephansen, Karen; Mattebjerg, Maria Ahlm; Wattjes, Jasper

2015-01-01

Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged...... fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta...

Endoplasmic Reticulum Stress and Obesity.

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Yilmaz, Erkan

2017-01-01

In recent years, the world has seen an alarming increase in obesity and closely associated with insulin resistance which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) play in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably other causes for obesity-related insulin resistance and inflammation. One of these appears to be endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.

Endoplasmic reticulum stress in lung disease

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Stefan J. Marciniak

2017-06-01

Full Text Available Exposure to inhaled pollutants, including fine particulates and cigarette smoke is a major cause of lung disease in Europe. While it is established that inhaled pollutants have devastating effects on the genome, it is now recognised that additional effects on protein folding also drive the development of lung disease. Protein misfolding in the endoplasmic reticulum affects the pathogenesis of many diseases, ranging from pulmonary fibrosis to cancer. It is therefore important to understand how cells respond to endoplasmic reticulum stress and how this affects pulmonary tissues in disease. These insights may offer opportunities to manipulate such endoplasmic reticulum stress pathways and thereby cure lung disease.

Localization and in-vivo characterization of thapsia garganica CYP76AE2 indicates a role in thapsigargin biosynthesis

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Martinez-Swatson, Karen Agatha; Rasmussen, Silas Anselm

2017-01-01

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+ -ATPase calcium pump in mammals and is of industrial importance as the active...

Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

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Smirnov Vladimir N

2002-10-01

Full Text Available Abstract Background Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown. Results We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation. Conclusions The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

The structural basis of calcium transport by the calcium pump

DEFF Research Database (Denmark)

Olesen, Claus; Picard, Martin; Winther, Anne-Marie Lund

2007-01-01

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding,...

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

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Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

2012-10-15

Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

Membrane defect in procine malignant hyperthermia

International Nuclear Information System (INIS)

O'Brien, P.J.

1985-01-01

Malignant hyperthermia (MH) has been proposed to result from abnormal calcium-homeostasis in skeletal muscle. This study tested the hypothesis that calcium-sequestration or calcium-release by sarcoplasmic reticulum was abnormal in MH-susceptible swine. A heavy sarcoplasmic reticulum fraction (HSR), enriched in terminal cisternae, was isolated from MH and control muscle using differential and density-gradient centrifugation. Calcium transport was studied using 45 Ca radioisotope and Millipore filtration. Enzymatic activities, cholesterol, phospholipid, and protein composition were determined using spectrophotometric techniques and polyacrylamide gel electrophoresis. Properties of calcium-sequestration by MH and control HSR were indistinguishable, although Ca 2+ -ATPase and calsequestrin content were 100% increased in MH HSR. However when muscle homogenate pH was decreased due to MH, calcium-uptake activity was depressed to <5% of control values. Results of this study indicate a model for the etiopathogenesis of MH, and for the inheritance and diagnosis of susceptibility to MH. Malignant hyperthermia is initiated due to a hypersensitive HSR calcium-release mechanism and propagated by a loss of calcium-sequestering function as acidosis develops. Susceptibility is inherited in an autosomal, codominant pattern and may be diagnosed most definitively and sensitively on the basis of calcium-release sensitivity-tests, performed on isolated HSR

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

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Linde, Cristina I.; Karashima, Eiji; Raina, Hema; Zulian, Alessandra; Wier, Withrow G.; Hamlyn, John M.; Ferrari, Patrizia; Blaustein, Mordecai P.

2012-01-01

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1–100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives.

NARCIS (Netherlands)

Treves, S.; Jungbluth, H.; Voermans, N.C.; Muntoni, F.; Zorzato, F.

2017-01-01

The physiological process by which Ca2+ is released from the sarcoplasmic reticulum is called excitation-contraction coupling; it is initiated by an action potential which travels deep into the muscle fiber where it is sensed by the dihydropyridine receptor, a voltage sensing L-type Ca2+channel

[3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

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Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

2002-06-14

Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

Regulatory effects of phospholamban on cardiac sarcoplasmic reticulum function

International Nuclear Information System (INIS)

Kim, Hae Won.

1989-01-01

In this thesis, the author reports the effect of phospholamban on: (a) Ca 2+ release by cardiac SR and (b) the Ca 2+ -ATPase activity in a purified reconstituted system. Phosphorylation of phospholamban by Ca 2+ · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca 2+ release from cardiac SR vesicles loaded under passive conditions and on the apparent 45 Ca 2+ - 40 Ca 2+ exchange from cardiac SR vesicles loaded under active conditions. us, it appears that Ca 2+ · calmodulin-dependent phosphorylation of phospholamban is not involved in the regulation of Ca 2+ release and 45 Ca 2+-40 Ca 2+ exchange. To determine the molecular mechanism by which phospholamban regulates the Ca 2+ pump, a reconstituted system was developed, using a freeze-thaw sonication procedure. The Ca 2+ -ATPase was purified by a method which yields an active enzyme preparation essentially free of phospholamban. The best rates of Ca 2+ uptake were obtained when cholate and phosphatidylcholine (PC) were used at a ratio of cholate/PC/Ca 2 + -ATPase of 2/80/1. The maximal rates of Ca 2+ Uptake were 700 nmol/min/mg reconstituted vesicles compared to 800 nmol/min/mg SR vesicles. The EC 50 values for Ca 2+ were 0.05 μM for both Ca 2+ uptake and Ca 2+ -ATPase activity in the reconstituted vesicles compared to 0.63 μM Ca 2+ in native SR vesicles. To determine the effect of phospholamban on the Ca + -ATPase activity in the reconstituted vesicles, purified phospholamban was added to the cholate/Ca 2+ -ATPase mixture prior to combining it with liposomes

Endoplasmic reticulum stress in wake-active neurons progresses with aging.

Science.gov (United States)

Naidoo, Nirinjini; Zhu, Jingxu; Zhu, Yan; Fenik, Polina; Lian, Jie; Galante, Ray; Veasey, Sigrid

2011-08-01

Fragmentation of wakefulness and sleep are expected outcomes of advanced aging. We hypothesize that wake neurons develop endoplasmic reticulum dyshomeostasis with aging, in parallel with impaired wakefulness. In this series of experiments, we sought to more fully characterize age-related changes in wakefulness and then, in relevant wake neuronal populations, explore functionality and endoplasmic reticulum homeostasis. We report that old mice show greater sleep/wake transitions in the active period with markedly shortened wake periods, shortened latencies to sleep, and less wake time in the subjective day in response to a novel social encounter. Consistent with sleep/wake instability and reduced social encounter wakefulness, orexinergic and noradrenergic wake neurons in aged mice show reduced c-fos response to wakefulness and endoplasmic reticulum dyshomeostasis with increased nuclear translocation of CHOP and GADD34. We have identified an age-related unfolded protein response injury to and dysfunction of wake neurons. It is anticipated that these changes contribute to sleep/wake fragmentation and cognitive impairment in aging. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Immune responsiveness and incidence of reticulum cell sarcoma in long-term syngeneic radiation chimeras

International Nuclear Information System (INIS)

Adorini, L.; Gorini, G.; Covelli, V.; Ballardin, E.; di Michele, A.; Bassani, B.; Metalli, P.; Doria, G.

1976-01-01

Long-term syngeneic radiation chimeras displayed a very low incidence of reticulum cell sarcoma as compared with control mice. Immune reactivity of these animals was studied in vivo by anti-dinitrophenyl antibody titer and affinity and in vitro by mitotic responsiveness to phytohemagglutinin, concanavalin A and lipopolysaccharide. Antibody titer and affinity as well as the response to T lectins were found to be increased in chimeras. These results were attributed to increased function of mature T2 cells, which could explain the reduced incidence of reticulum cell sarcoma in chimeras

Location matters: the endoplasmic reticulum and protein trafficking in dendrites

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Omar A Ramírez

2011-01-01

Full Text Available Neurons are highly polarized, but the trafficking mechanisms that operate in these cells and the topological organization of their secretory organelles are still poorly understood. Particularly incipient is our knowledge of the role of the neuronal endoplasmic reticulum. Here we review the current understanding of the endoplasmic reticulum in neurons, its structure, composition, dendritic distribution and dynamics. We also focus on the trafficking of proteins through the dendritic endoplasmic reticulum, emphasizing the relevance of transport, retention, assembly of multi-subunit protein complexes and export. We additionally discuss the roles of the dendritic endoplasmic reticulum in synaptic plasticity.

Morphological and Biochemical Characterization of Bovine Congenital Psudomyotonia

OpenAIRE

Dorotea, Tiziano

2015-01-01

The Ca2+-ATPase of sarco(endo)plasmic reticulum (SERCA) is a protein of about 110 kDa member of the P-type ATPases family. SERCA pumps utilize the energy derived from the hydrolysis of a molecule of ATP to transport two Ca2+ ions across the Sarcoplasmic Reticulum (SR) membrane to decrease the Ca2+ concentration in the cytosol. SERCA isoform 1a (SERCA1a) is the mainly expressed isoform in adult fast-twitch muscle fibre and it is both structurally and functionally the best characterized member ...

Effects of ginger extract on smooth muscle activity of sheep reticulum and rumen

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Amin Mamaghani

2013-06-01

Full Text Available Reticulorumen hypomotility leads to the impaired physiologic functions of the digestive tract. Prokinetic action of ginger has been demonstrated in the laboratory animals and human. The aim of this study was to evaluate the effect of hydroalcoholic extract of ginger on contraction and motility of reticulum and rumen of ruminants. Collected samples of reticulum and rumen from eight sheep were investigated in vitro. The extract at the concentration of 0.1 and 1.0 mg L-1 had no effect on any preparations. Contraction of reticulum and rumen preparations was occurred at 10.0 and 100 mg L-1 concentrations (p < 0.05. Concentration of 1000 mg L-1 caused a relaxation in preparations contracted with 10.0 and 100 mg L-1. Likewise, the concentration of 1000 mg L-1 significantly (p < 0.05 inhibited ACh-induced contraction in both tissues. Six sheep were involved in electromyographic study. Administration of 40 mg kg-1 of the extract increased the overall frequency of contractions of the reticulum and rumen at the subsequent three days with the prominent increase at the second day (p < 0.05. Results of in vitro study indicated that hydroalcoholic extract of ginger contained spasmogenic and spasmolytic constituents. The results in vivo study represented evidences that the extract may have stimulant effect on reticulorumen motility in 40 mg kg-1 concentration.

Effects of chronic administration of clenbuterol on contractile properties and calcium homeostasis in rat extensor digitorum longus muscle.

Science.gov (United States)

Sirvent, Pascal; Douillard, Aymerick; Galbes, Olivier; Ramonatxo, Christelle; Py, Guillaume; Candau, Robin; Lacampagne, Alain

2014-01-01

Clenbuterol, a β2-agonist, induces skeletal muscle hypertrophy and a shift from slow-oxidative to fast-glycolytic muscle fiber type profile. However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. As the intracellular Ca2+ concentration must be finely regulated in many cellular processes, the aim of this study was to investigate the effects of chronic clenbuterol treatment on force, fatigue, intracellular calcium (Ca2+) homeostasis and Ca2+-dependent proteolysis in fast-twitch skeletal muscles (the extensor digitorum longus, EDL, muscle), as they are more sensitive to clenbuterol-induced hypertrophy. Male Wistar rats were chronically treated with 4 mg.kg-1 clenbuterol or saline vehicle (controls) for 21 days. Confocal microscopy was used to evaluate sarcoplasmic reticulum Ca2+ load, Ca2+-transient amplitude and Ca2+ spark properties. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle. Compared to control animals, clenbuterol treatment decreased Ca2+-transient amplitude, Ca2+ spark amplitude and frequency and the sarcoplasmic reticulum Ca2+ load was markedly reduced. Conversely, calpain activity was increased by clenbuterol chronic treatment. These results indicate that chronic treatment with clenbuterol impairs Ca2+ homeostasis and this could contribute to the remodeling and functional impairment of fast-twitch skeletal muscle.

C[sub 10]-O[sub eq]-N-(4-azido-5-[sup 125]iodo salicyloyl)-[beta]-alanyl-[beta] alanyl ryanodine (Az-[beta]AR), a novel photo-affinity ligand for the ryanodine binding site

Energy Technology Data Exchange (ETDEWEB)

Bidasee, K.R.; Besch, H.R. Jr.; Kwon, Sangyeol; Emmick, J.T.; Besch, K.T.; Gerzon, Koert; Humerickhouse, R.A. (Indiana Univ., Indianapolis, IN (United States). School of Medicine)

1994-01-01

A high affinity, photoactivatable, radio-iodinated ligand for the ryanodine binding site(s) of the sarcoplasmic reticulum calcium-release channel, C[sub 10]-O[sub e]-N-(4-azido-5-[sup 125]iodo salicyloyl)-[beta]-alanyl-[beta]-alanyl ryanodine (Az-[beta]AR), was synthesized at a specific activity of 1400mCi/mmol. (Author).

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA) activity

International Nuclear Information System (INIS)

Benaim, Gustavo; Pimentel, Adriana A.; Felibertt, Pimali; Mayora, Adriana; Colman, Laura; Sojo, Felipe; Rojas, Héctor; De Sanctis, Juan B.

2016-01-01

The increase in the intracellular Ca"2"+ concentration ([Ca"2"+]_i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca"2"+]_i by inhibiting the sarco(endo)plasmic reticulum Ca"2"+-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca"2"+ pump. The results showed that addition of sphingosine produced a release of Ca"2"+ from the endoplasmic reticulum followed by a Ca"2"+ entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca"2"+]_I in mammalian cells.

Ghrelin Ameliorates Asthma by Inhibiting Endoplasmic Reticulum Stress.

Science.gov (United States)

Fu, Tian; Wang, Lei; Zeng, Qingdi; Zhang, Yan; Sheng, Baowei; Han, Liping

2017-12-01

This study aimed to confirm the ameliorative effect of ghrelin on asthma and investigate its mechanism. The murine model of asthma was induced by ovalbumin (OVA) treatment and assessed by histological pathology and airway responsiveness to methacholine. The total and differential leukocytes were counted. Tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 levels in bronchoalveolar lavage fluid were quantified by commercial kits. The protein levels in pulmonary tissues were measured by Western blot analysis. Ghrelin ameliorated the histological pathology and airway hyperresponsiveness in the OVA-induced asthmatic mouse model. Consistently, OVA-increased total and differential leukocytes and levels of tumor necrosis factor α, interferon γ, interleukin-5 and interleukin-13 in bronchoalveolar lavage fluid were significantly attenuated by ghrelin. Ghrelin prevented the increased protein levels of the endoplasmic reticulum stress markers glucose regulated protein 78 and CCAAT/enhancer binding protein homologous protein and reversed the reduced levels of p-Akt in asthmatic mice. Ghrelin might prevent endoplasmic reticulum stress activation by stimulating the Akt signaling pathway, which attenuated inflammation and ameliorated asthma in mice. Ghrelin might be a new target for asthma therapy. Copyright © 2017. Published by Elsevier Inc.

Ixeris dentata Extract Increases Salivary Secretion through the Regulation of Endoplasmic Reticulum Stress in a Diabetes-Induced Xerostomia Rat Model.

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Bhattarai, Kashi Raj; Lee, Hwa-Young; Kim, Seung-Hyun; Kim, Hyung-Ryong; Chae, Han-Jung

2018-04-02

This study aimed to investigate the molecular mechanism of diabetes mellitus (DM)-induced dry mouth and an application of natural products from Ixeris dentata (IXD), a recently suggested regulator of amylase secretion in salivary cells. Vehicle-treated or diabetic rats were orally treated with either water or an IXD extract for 10 days to observe the effect on salivary flow. We found that the IXD extract increased aquaporin 5 (AQP5) and alpha-amylase protein expression in the submandibular gland along with salivary flow rate. Similarly, the IXD extract and its purified compound increased amylase secretion in high glucose-exposed human salivary gland cells. Furthermore, increased endoplasmic reticulum stress response in the submandibular gland of diabetic rats was inhibited by treatment with the IXD extract, suggesting that IXD extract treatment improves the ER environment by increasing the protein folding capacity. Thus, pharmacological treatment with the IXD extract is suggested to relieve DM-induced dry mouth symptoms.

Ixeris dentata Extract Increases Salivary Secretion through the Regulation of Endoplasmic Reticulum Stress in a Diabetes-Induced Xerostomia Rat Model

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Kashi Raj Bhattarai

2018-04-01

Full Text Available This study aimed to investigate the molecular mechanism of diabetes mellitus (DM-induced dry mouth and an application of natural products from Ixeris dentata (IXD, a recently suggested regulator of amylase secretion in salivary cells. Vehicle-treated or diabetic rats were orally treated with either water or an IXD extract for 10 days to observe the effect on salivary flow. We found that the IXD extract increased aquaporin 5 (AQP5 and alpha-amylase protein expression in the submandibular gland along with salivary flow rate. Similarly, the IXD extract and its purified compound increased amylase secretion in high glucose-exposed human salivary gland cells. Furthermore, increased endoplasmic reticulum stress response in the submandibular gland of diabetic rats was inhibited by treatment with the IXD extract, suggesting that IXD extract treatment improves the ER environment by increasing the protein folding capacity. Thus, pharmacological treatment with the IXD extract is suggested to relieve DM-induced dry mouth symptoms.

A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

NARCIS (Netherlands)

Koenderink, J.B.; Swarts, H.G.P.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

2004-01-01

Homology modeling of gastric H, K-ATPase based on the E-2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791)

A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase.

NARCIS (Netherlands)

Koenderink, J.B.; Swarts, H.G.P.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

2004-01-01

Homology modeling of gastric H,K-ATPase based on the E2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E2 form-specific salt bridge between Glu820 (M6) and Lys791 (M5).

Hydrogen peroxide increases depolarization-induced contraction of mechanically skinned slow twitch fibres from rat skeletal muscles.

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Plant, David R; Lynch, Gordon S; Williams, David A

2002-03-15

The effect of exogenous hydrogen peroxide (H(2)O(2)) on excitation-contraction (E-C) coupling and sarcoplasmic reticulum (SR) function was compared in mechanically skinned slow twitch fibres (prepared from the soleus muscles) and fast twitch fibres (prepared from the extensor digitorum longus; EDL muscles) of adult rats. Equilibration (5 min) with 1 mM H(2)O(2) diminished the ability of the Ca(2+)-depleted SR to reload Ca(2+) in both slow (P fast twitch fibres (P fast twitch fibres by 24 +/- 5 % (P slow twitch fibres. Treatment with 1 mM H(2)O(2) also increased the peak force of low [caffeine] contracture by approximately 45% in both fibre types compared to control (P slow twitch fibres, compared to control (no H(2)O(2); P fast twitch fibres was not altered by 1 mM H(2)O(2) treatment. Equilibration with 5 mM H(2)O(2) induced a spontaneous force response in both slow and fast twitch fibres, which could be partly reversed by 2 min treatment with 10 mM DTT. Peak DICR was also increased approximately 40% by 5 mM H(2)O(2) in slow twitch fibres compared to control (no H(2)O(2); P slow but not fast twitch fibres. The increase in depolarization-induced contraction in slow twitch fibres might be mediated by an increased SR Ca(2+) release during contraction and/or an increase in Ca(2+) sensitivity.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

Science.gov (United States)

Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

1H nuclear magnetic resonance studies of sarcoplasmic oxygenation in the red cell-perfused rat heart

OpenAIRE

Jelicks, L.A.; Wittenberg, B.A.

1995-01-01

The proximal histidine N delta H proton of deoxymyoglobin experiences a large hyperfine shift resulting in its 1H nuclear magnetic resonance (NMR) signal appearing at approximately 76 ppm (at 35 degrees C), downfield of the diamagnetic spectral region. 1H NMR of this proton is used to monitor sarcoplasmic oxygen pressure in isolated perfused rat heart. This method monitors intracellular oxygenation in the whole heart and does not reflect oxygenation in a limited region. The deoxymyoglobin res...

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Institute of Scientific and Technical Information of China (English)

Liping Yang; Lemeng Wu; Dongmei Wang; Ying Li; Hongliang Dou; Mark OMTso; Zhizhong Ma

2013-01-01

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in-creased, which was associated with retinal ganglion celldeath in diabetic retinas. The C/ERB ho-mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in-dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu-ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Clofibric Acid Increases the Formation of Oleic Acid in Endoplasmic Reticulum of the Liver of Rats

OpenAIRE

広瀬, 明彦; 山崎, 研; 坂本, 武史; 須永, 克佳; 津田, 整; 光本, 篤史; 工藤, なをみ; 川嶋, 洋一

2011-01-01

The effects of 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) on the formation of oleic acid (18:1) from stearic acid (18:0) and utilization of the 18:1 formed for phosphatidylcholine (PC) formation in endoplasmic reticulum in the liver of rats were studied in vivo. [14C]18:0 was intravenously injected into control Wistar male rats and rats that had been fed on a diet containing 0.5% (w/w) clofibric acid for 7 days; and the distribution of radiolabeled fatty acids among subcellul...

Prediction of endoplasmic reticulum resident proteins using fragmented amino acid composition and support vector machine

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Ravindra Kumar

2017-09-01

Full Text Available Background The endoplasmic reticulum plays an important role in many cellular processes, which includes protein synthesis, folding and post-translational processing of newly synthesized proteins. It is also the site for quality control of misfolded proteins and entry point of extracellular proteins to the secretory pathway. Hence at any given point of time, endoplasmic reticulum contains two different cohorts of proteins, (i proteins involved in endoplasmic reticulum-specific function, which reside in the lumen of the endoplasmic reticulum, called as endoplasmic reticulum resident proteins and (ii proteins which are in process of moving to the extracellular space. Thus, endoplasmic reticulum resident proteins must somehow be distinguished from newly synthesized secretory proteins, which pass through the endoplasmic reticulum on their way out of the cell. Approximately only 50% of the proteins used in this study as training data had endoplasmic reticulum retention signal, which shows that these signals are not essentially present in all endoplasmic reticulum resident proteins. This also strongly indicates the role of additional factors in retention of endoplasmic reticulum-specific proteins inside the endoplasmic reticulum. Methods This is a support vector machine based method, where we had used different forms of protein features as inputs for support vector machine to develop the prediction models. During training leave-one-out approach of cross-validation was used. Maximum performance was obtained with a combination of amino acid compositions of different part of proteins. Results In this study, we have reported a novel support vector machine based method for predicting endoplasmic reticulum resident proteins, named as ERPred. During training we achieved a maximum accuracy of 81.42% with leave-one-out approach of cross-validation. When evaluated on independent dataset, ERPred did prediction with sensitivity of 72.31% and specificity of 83

FGF-23 dysregulates calcium homeostasis and electrophysiological properties in HL-1 atrial cells.

Science.gov (United States)

Kao, Yu-Hsun; Chen, Yao-Chang; Lin, Yung-Kuo; Shiu, Rong-Jie; Chao, Tze-Fan; Chen, Shih-Ann; Chen, Yi-Jen

2014-08-01

Fibroblast growth factor (FGF)-23 is a key regulator of phosphate homeostasis. Higher FGF-23 levels are correlated with poor outcomes in cardiovascular diseases. FGF-23 can produce cardiac hypertrophy and increase intracellular calcium, which can change cardiac electrical activity. However, it is not clear whether FGF-23 possesses arrhythmogenic potential through calcium dysregulation. Therefore, the purposes of this study were to evaluate the electrophysiological effects of FGF-23 and identify the underlying mechanisms. Patch clamp, confocal microscope with Fluo-4 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis and calcium regulatory proteins in HL-1 atrial myocytes with and without FGF-23 (10 and 25 ng/mL) incubation for 24 h. FGF-23 (25 ng/mL) increased L-type calcium currents, calcium transient and sarcoplasmic reticulum Ca(2+) contents in HL-1 cells. FGF-23 (25 ng/mL)-treated cells (n = 14) had greater incidences (57%, 17% and 15%, P calcium/calmodulin-dependent protein kinase IIδ and phospholamban (PLB) at threonine 17 but had similar phosphorylation extents of PLB at serine 16, total PLB and sarcoplasmic reticulum Ca(2+) -ATPase protein. Moreover, the FGF receptor inhibitor (PD173074, 10 nM), calmodulin inhibitor (W7, 5 μM) and phospholipase C inhibitor (U73122, 1 μM) attenuated the effects of FGF-23 on calcium/calmodulin-dependent protein kinase II phosphorylation. FGF-23 increases HL-1 cells arrhythmogenesis with calcium dysregulation through modulating calcium-handling proteins. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

In Vivo and In Silico Investigation Into Mechanisms of Frequency Dependence of Repolarization Alternans in Human Ventricular Cardiomyocytes.

Science.gov (United States)

Zhou, Xin; Bueno-Orovio, Alfonso; Orini, Michele; Hanson, Ben; Hayward, Martin; Taggart, Peter; Lambiase, Pier D; Burrage, Kevin; Rodriguez, Blanca

2016-01-22

Repolarization alternans (RA) are associated with arrhythmogenesis. Animal studies have revealed potential mechanisms, but human-focused studies are needed. RA generation and frequency dependence may be determined by cell-to-cell variability in protein expression, which is regulated by genetic and external factors. To characterize in vivo RA in human and to investigate in silico using human models, the ionic mechanisms underlying the frequency-dependent differences in RA behavior identified in vivo. In vivo electrograms were acquired at 240 sites covering the epicardium of 41 patients at 6 cycle lengths (600-350 ms). In silico investigations were conducted using a population of biophysically detailed human models incorporating variability in protein expression and calibrated using in vivo recordings. Both in silico and in vivo, 2 types of RA were identified, with Fork- and Eye-type restitution curves, based on RA persistence or disappearance, respectively, at fast pacing rates. In silico simulations show that RA are strongly correlated with fluctuations in sarcoplasmic reticulum calcium, because of strong release and weak reuptake. Large L-type calcium current conductance is responsible for RA disappearance at fast frequencies in Eye-type (30% larger in Eye-type versus Fork-type; Psilico, 2 types of RA are identified, with RA persistence/disappearance as frequency increases. In silico, L-type calcium current and Na(+)/Ca(2+) exchanger current determine RA human cell-to-cell differences through intracellular and sarcoplasmic reticulum calcium regulation. © 2015 The Authors.

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability*

Science.gov (United States)

Liu, Yiting; Lee, Sang Y.; Neely, Elizabeth; Nandar, Wint; Moyo, Mthabisi; Simmons, Zachary; Connor, James R.

2011-01-01

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability. PMID:21349849

4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.

Science.gov (United States)

Liu, Liangming; Wu, Huiling; Zang, JiaTao; Yang, Guangming; Zhu, Yu; Wu, Yue; Chen, Xiangyun; Lan, Dan; Li, Tao

2016-08-01

Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. Laboratory investigation. State Key Laboratory of Trauma, Burns and Combined Injury. Sprague-Dawley rats. Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. Anti

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

Science.gov (United States)

Macková, Katarina; Zahradníková, Alexandra; Hoťka, Matej; Hoffmannová, Barbora; Zahradník, Ivan; Zahradníková, Alexandra

2017-12-01

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress

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Voin Petrovic

2018-04-01

Full Text Available The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE. We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER stress.

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells

Science.gov (United States)

Yu, Kyeong-Nam; Chang, Seung-Hee; Park, Soo Jin; Lim, Joohyun; Lee, Jinkyu; Yoon, Tae-Jong; Kim, Jun-Sung; Cho, Myung-Haing

2015-01-01

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 μg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application. PMID:26121477

Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity

Energy Technology Data Exchange (ETDEWEB)

Benaim, Gustavo, E-mail: gbenaim@idea.gob.ve [Instituto de Estudios Avanzados (IDEA), Caracas (Venezuela, Bolivarian Republic of); Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Pimentel, Adriana A., E-mail: adriana.pimentel@ucv.ve [Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Felibertt, Pimali [Facultad de Ciencias, Universidad de Carabobo, Valencia (Venezuela, Bolivarian Republic of); Mayora, Adriana [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Colman, Laura [Instituto Pasteur de Montevideo, Montevideo (Uruguay); Sojo, Felipe [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Rojas, Héctor [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas (Venezuela, Bolivarian Republic of); De Sanctis, Juan B. [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of)

2016-04-29

The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

Science.gov (United States)

Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

2013-11-01

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The involvement of SMILE/TMTC3 in endoplasmic reticulum stress response.

Directory of Open Access Journals (Sweden)

Maud Racapé

Full Text Available The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER. In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

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Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

2016-04-20

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Soybean oil increases SERCA2a expression and left ventricular contractility in rats without change in arterial blood pressure

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Vassallo Dalton

2010-05-01

Full Text Available Abstract Background Our aim was to evaluate the effects of soybean oil treatment for 15 days on arterial and ventricular pressure, myocardial mechanics and proteins involved in calcium handling. Methods Wistar rats were divided in two groups receiving 100 μL of soybean oil (SB or saline (CT i.m. for 15 days. Ventricular performance was analyzed in male 12-weeks old Wistar rats by measuring left ventricle diastolic and systolic pressure in isolated perfused hearts according to the Langendorff technique. Protein expression was measured by Western blot analysis. Results Systolic and diastolic arterial pressures did not differ between CT and SB rats. However, heart rate was reduced in the SB group. In the perfused hearts, left ventricular isovolumetric systolic pressure was higher in the SB hearts. The inotropic response to extracellular Ca2+ and isoproterenol was higher in the soybean-treated animals than in the control group. Myosin ATPase and Na+-K+ATPase activities, the expression of sarcoplasmic reticulum calcium pump (SERCA2a and sodium calcium exchanger (NCX were increased in the SB group. Although the phosfolamban (PLB expression did not change, its phosphorylation at Ser16 was reduced while the SERCA2a/PLB ratio was increased. Conclusions In summary, soybean treatment for 15 days in rats increases the left ventricular performance without affecting arterial blood pressure. These changes might be associated with an increase in the myosin ATPase activity and SERCA2a expression.

The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

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Chao de la Barca, Juan Manuel; Simard, Gilles; Amati-Bonneau, Patrizia; Safiedeen, Zainab; Prunier-Mirebeau, Delphine; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Gueguen, Naïg; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Ferré, Marc; Bris, Céline; Kouassi Nzoughet, Judith; Bocca, Cinzia; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Lenaers, Guy; Martinez, M Carmen; Procaccio, Vincent; Reynier, Pascal

2016-11-01

Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as

Clofibric acid increases the formation of oleic acid in endoplasmic reticulum of the liver of rats.

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Hirose, Akihiko; Yamazaki, Tohru; Sakamoto, Takeshi; Sunaga, Katsuyoshi; Tsuda, Tadashi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2011-01-01

The effects of 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) on the formation of oleic acid (18:1) from stearic acid (18:0) and utilization of the 18:1 formed for phosphatidylcholine (PC) formation in endoplasmic reticulum in the liver of rats were studied in vivo. [¹⁴C]18:0 was intravenously injected into control Wistar male rats and rats that had been fed on a diet containing 0.5% (w/w) clofibric acid for 7 days; and the distribution of radiolabeled fatty acids among subcellular organelles, microsomes, peroxisomes, and mitochondria, was estimated on the basis of correction utilizing the yields from homogenates of marker enzymes for these organelles. The radioactivity was mostly localized in microsomes and the radiolabeled fatty acids present in microsomes were significantly increased by the treatment of rats with clofibric acid. The formation of radiolabeled 18:1 in microsomes markedly increased and incorporations of the formed [¹⁴C]18:1 into PC and phosphatidylethanolamine in microsomes were augmented in response to clofibric acid. The [¹⁴C]18:1 incorporated into PC was mostly located at the C-2 position, but not the C-1 position, of PC, and the radioactivity in 18:1 at the C-2 position of PC was strikingly increased by clofibric acid. These results obtained from the in vivo experiments directly link the findings that clofibric acid treatment induces microsomal stearoyl-CoA desaturase and 1-acylglycerophosphocholine acyltransferase in the liver and the findings that the treatment with the drug elevated absolute mass and mass proportion of 18:1 at the C-2 position, but not the C-1 position, of PC in the liver together.

Recovery of Action Potentials and Twitches after K-contractures in Frog Skeletal Muscle(Physiology)

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Atsuko, Suzuki; Ibuki, Shirakawa; Kazunari, Noguchi; Hirohiko, Kishi; Haruo, Sugi; Department of Physiology, School of Medicine, Teikyo University:(Present office)Department of Physical Therapy, Health Science University; Department of Physiology, School of Medicine, Teikyo University; Department of Physiology, School of Medicine, Teikyo University; Department of Physiology, School of Medicine, Teikyo University; Department of Physiology, School of Medicine, Teikyo University

2004-01-01

To give information about intracellular Ca^ translocation during and after K-contractures in vertebrate skeletal muscle fibers, we examined recovery of action potentials and twitches after interruption and spontaneous relaxation of K-contractures at low temperature (3℃) that greatly reduced the rate of Ca^ reuptake by the sarcoplasmic reticulum. On membrane repolarization interrupting K-contractures, the amplitude of both action potentials and twitches recovered quickly, while the falling pha...

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels

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Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-01-01

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca2+ ATPase (SERCA), including the widely expres...

Acute exposure to lead increases myocardial contractility independent of hypertension development

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Fioresi, M. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Departamento de Enfermagem, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Furieri, L.B.; Simões, M.R.; Ribeiro, R.F. Junior; Meira, E.F.; Fernandes, A.A.; Stefanon, I. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Vassallo, D.V. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Centro de Ciências da Saúde de Vitória, Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, Vitória, ES (Brazil)

2013-02-01

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetate iv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na{sup +},K{sup +}-ATPase and myosin Ca{sup 2+}-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2 vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13 vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na{sup +},K{sup +}-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Acute exposure to lead increases myocardial contractility independent of hypertension development

International Nuclear Information System (INIS)

Fioresi, M.; Furieri, L.B.; Simões, M.R.; Ribeiro, R.F. Junior; Meira, E.F.; Fernandes, A.A.; Stefanon, I.; Vassallo, D.V.

2013-01-01

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetate iv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na + ,K + -ATPase and myosin Ca 2+ -ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2 vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13 vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na + ,K + -ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension

Endurance training in the spontaneously hypertensive rat: conversion of pathological into physiological cardiac hypertrophy.

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Garciarena, Carolina D; Pinilla, Oscar A; Nolly, Mariela B; Laguens, Ruben P; Escudero, Eduardo M; Cingolani, Horacio E; Ennis, Irene L

2009-04-01

The effect of endurance training (swimming 90 min/d for 5 days a week for 60 days) on cardiac hypertrophy was investigated in the spontaneously hypertensive rat (SHR). Sedentary SHRs (SHR-Cs) and normotensive Wistar rats were used as controls. Exercise training enhanced myocardial hypertrophy assessed by left ventricular weight/tibial length (228+/-7 versus 251+/-5 mg/cm in SHR-Cs and exercised SHRs [SHR-Es], respectively). Myocyte cross-sectional area increased approximately 40%, collagen volume fraction decreased approximately 50%, and capillary density increased approximately 45% in SHR-Es compared with SHR-Cs. The mRNA abundance of atrial natriuretic factor and myosin light chain 2 was decreased by the swimming routine (100+/-19% versus 41+/-10% and 100+/-8% versus 61+/-9% for atrial natriuretic factor and myosin light chain 2 in SHR-Cs and SHR-Es, respectively). The expression of sarcoplasmic reticulum Ca(2+) pump was significantly augmented, whereas that of Na(+)/Ca(2+) exchanger was unchanged (93+/-7% versus 167+/-8% and 158+/-13% versus 157+/-7%, sarcoplasmic reticulum Ca(2+) pump and Na(+)/Ca(2+) exchanger in SHR-Cs and SHR-Es, respectively; PEndurance training inhibited apoptosis, as reflected by a decrease in caspase 3 activation and poly(ADP-ribose) polymerase-1 cleavage, and normalized calcineurin activity without inducing significant changes in the phosphatidylinositol 3-kinase/Akt pathway. The swimming routine improved midventricular shortening determined by echocardiography (32.4+/-0.9% versus 36.9+/-1.1% in SHR-Cs and SHR-Es, respectively; Pendurance training to convert pathological into physiological hypertrophy improving cardiac performance. The reduction of myocardial fibrosis and calcineurin activity plus the increase in capillary density represent factors to be considered in determining this beneficial effect.

Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress

International Nuclear Information System (INIS)

Faria, Gisele; Cardoso, Cristina R.B.; Larson, Roy E.; Silva, Joao S.; Rossi, Marcos A.

2009-01-01

Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker of activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.

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Jintao Zhang

Full Text Available Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.Human colorectal cancer cell lines (HCT-116 and HT-29 were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining, and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II, beclin-1, and autophagocytosis-associated protein (Atg3. The autophagy inhibitors 3-methyladenine (3-MA and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin and genetic

Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells.

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Chini, Corryn E; Fisher, Gregory L; Johnson, Ben; Tamkun, Michael M; Kraft, Mary L

2018-02-26

Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX ® , which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS 2 ) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS 2 imaging of selected ions in parallel with the precursor ion (MS 1 ) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

Endoplasmic Reticulum Stress and Associated ROS

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Hafiz Maher Ali Zeeshan

2016-03-01

Full Text Available The endoplasmic reticulum (ER is a fascinating network of tubules through which secretory and transmembrane proteins enter unfolded and exit as either folded or misfolded proteins, after which they are directed either toward other organelles or to degradation, respectively. The ER redox environment dictates the fate of entering proteins, and the level of redox signaling mediators modulates the level of reactive oxygen species (ROS. Accumulating evidence suggests the interrelation of ER stress and ROS with redox signaling mediators such as protein disulfide isomerase (PDI-endoplasmic reticulum oxidoreductin (ERO-1, glutathione (GSH/glutathione disuphide (GSSG, NADPH oxidase 4 (Nox4, NADPH-P450 reductase (NPR, and calcium. Here, we reviewed persistent ER stress and protein misfolding-initiated ROS cascades and their significant roles in the pathogenesis of multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases.

Effect of endoplasmic reticulum stress on the response of HeLa cells to carbon ion radiation

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Xia Jiefang; Wang Zhuanzi; Wei Wei; Dang Bingrong; Li Wenjian

2015-01-01

To investigate the effect of endoplasmic reticulum stress on HeLa cells to "1"2C"6"+ ion irradiation, HeLa cells were pretreated with 2.5 mmol/L dithiothreitol and irradiated by "1"2C"6"+ ions with different doses. The results showed that, compared with IR alone, dithiothreitol combined with carbon ion irradiation caused HeLa cell survival decreased, and the apoptosis increased. Moreover, dithiothreitol and carbon ion radiation combination treatment led to a significant increase of G_2/M phase, and autophagy was activated obviously in combination treatment group. The results imply that continuous endoplasmic reticulum stress can change the response of HeLa cells to "1"2C"6"+ irradiation, and dithiothreitol may affect HeLa cells through the autophagy cell death pathway. (authors)

Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

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Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

2015-05-01

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

Ryanodine receptor 1 and associated pathologies

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Fauré , Julien; Lunardi , Joël; Monnier , Nicole; Marty , Isabelle

2014-01-01

In skeletal muscle a rise in the cytosolic calcium concentration is the first trigger able to initiate the contraction of the sarcomere. Intracellular calcium levels are tightly controlled by channels and pumps, and it is not surprising that many inherited skeletal muscle disorders arise from mutations altering the players regulating calcium ions concentration (Betzenhauser et al., 2010). In this chapter, we will focus on the pathologies linked to the sarcoplasmic reticulum calcium channel-Ry...

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?

DEFF Research Database (Denmark)

Skagen, Peter Storgaard; Horn, T; Kruse, H A

2011-01-01

Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities...... in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in "bundles" and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae...... polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification....

Temperature dependence of cardiac sarcoplasmic reticulum and sarcolemma in the ventricle of catfish (Clarias gariepinus

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El-Sabry Abu-Amra

2015-10-01

The sarcolemmal Ca2+ contribution of activator Ca2+ was greater at a test temperature of 30 °C as assessed by verapamil. Whereas the SR-Ca2+ contribution was higher at 20 and 30 °C and a frequency rate of 0.2 and 0.4 Hz as assessed by caffeine and adrenaline, respectively. Bradykinin potentiating factor (BPF7 which was isolated from jelly fish (Cassiopea andromeda decreased the cardiac force developed at a frequency rate of 0.2 Hz and a temperature of 20 °C, whereas it increased the force developed at frequency rates of 0.2 and 0.4 Hz at 30 °C. These results indicate that BPF7 may act like verapamil in reducing the cardiac force through blocking the sarcolemmal Ca2+ channels at low temperature and like adrenaline in an increase of the cardiac force developed at warm temperature and the high frequency rate through stimulation of SR-Ca2+ activator. Therefore, this study indicates that the sarcolemmal Ca2+ influx and the SR-Ca2+ release contributors of activator Ca2+ for cardiac force development in the catfish heart were significantly greater at warm temperature and at the pacing frequency rates of 0.2 and 0.4 Hz as assessed by verapamil, adrenaline, caffeine and BPF7. However, the relative contribution of the sarcolemmal Ca2+ influx in the development of cardiac force in the catfish heart was greater than that of SR-Ca2+ release.

Selective modulation of endoplasmic reticulum stress markers in prostate cancer cells by a standardized mangosteen fruit extract.

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Gongbo Li

Full Text Available The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.

Selective modulation of endoplasmic reticulum stress markers in prostate cancer cells by a standardized mangosteen fruit extract.

Science.gov (United States)

Li, Gongbo; Petiwala, Sakina M; Pierce, Dana R; Nonn, Larisa; Johnson, Jeremy J

2013-01-01

The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.

Cholesterol Removal from Adult Skeletal Muscle impairs Excitation-Contraction Coupling and Aging reduces Caveolin-3 and alters the Expression of other Triadic Proteins

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Genaro eBarrientos

2015-04-01

Full Text Available Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not sarcoplasmic reticulum membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX activity and protein content of NOX2 subunits (p47phox and gp91phox, implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs excitation-contraction coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged

Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

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Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

2016-04-01

It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

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Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

2006-03-20

In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in association with its effects suppressing endoplasmic reticulum stress in db/db mice.

Science.gov (United States)

Wu, Yan-ju; Guo, Xin; Li, Chun-jun; Li, Dai-qing; Zhang, Jie; Yang, Yiping; Kong, Yan; Guo, Hang; Liu, De-min; Chen, Li-ming

2015-02-01

Vildagliptin promotes beta cell survival by inhibiting cell apoptosis. It has been suggested that chronic ER (endoplasmic reticulum) stress triggers beta cell apoptosis. The objective of the study is to explore whether the pro-survival effect of vildagliptin is associated with attenuation of endoplasmic reticulum stress in islets of db/db mice. Vildagliptin was orally administered to db/db mice for 6 weeks, followed by evaluation of beta cell apoptosis by caspase3 activity and TUNEL staining method. Endoplasmic reticulum stress markers were determined with quantitative RT-PCR, immunohistochemistry and immunoblot analysis. After 6 weeks of treatment, vildagliptin treatment increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44, Pvildagliptin treatment down-regulated several genes related to endoplasmic reticulum stress including TRIB3 (tribbles homolog 3) (15.9±0.4 vs. 33.3±1.7, ×10⁻³, PVildagliptin promoted beta cell survival in db/db mice in association with down-regulating markers of endoplasmic reticulum stress including TRIB3, ATF-4 as well as CHOP. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Protective role of antioxidant vitamin E and catechin on idarubicin-induced cardiotoxicity in rats

Directory of Open Access Journals (Sweden)

Kalender S.

2002-01-01

Full Text Available Idarubicin is an anthracycline antibiotic extensively used in acute leukemia. In the present study we investigated whether vitamin E and catechin can reduce the toxic effects of idarubicin. Vitamin E (200 IU kg-1 week-1, catechin (200 mg kg-1 week-1, idarubicin (5 mg kg-1 week-1, idarubicin + vitamin E (200 IU kg-1 week-1, and idarubicin + catechin (200 mg kg-1 week-1 combinations were given to male Sprague-Dawley rats weighing 210 to 230 g (N = 6/group. Idarubicin-treated animals exhibited a decrease in body and heart weight, a decrease in myocardial contractility, and changes in ECG parameters (P<0.01. Catechin + idarubicin- and vitamin E + idarubicin-treated groups exhibited similar alterations, but changes were attenuated in comparison to those in cardiac muscle of idarubicin-treated rats (P<0.05. Superoxide dismutase and catalase activity was reduced in the idarubicin-treated group (P<0.05. Glutathione peroxidase levels were decreased in the idarubicin-treated group (P<0.05 and reached maximum concentrations in the catechin- and catechin + idarubicin-treated groups compared to control (P<0.01. Malondialdehyde activity was decreased in the catechin + idarubicin-treated groups compared to control and increased in the other groups, reaching maximum concentrations in the vitamin E-treated group (P<0.01. In electron microscopy studies, swelling of the mitochondria and dilatation of the sarcoplasmic reticulum of myocytes were observed in the idarubicin-treated groups. In groups that were given idarubicin + vitamin E and idarubicin + catechin, the only morphological change was a weak dilatation of the sarcoplasmic reticulum. We conclude that catechin and vitamin E significantly reduce idarubicin-induced cardiotoxicity in rats.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

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Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

2016-09-20

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

International Nuclear Information System (INIS)

Luo, Ying; Li, Shu-Jun; Yang, Jian; Qiu, Yuan-Zhen; Chen, Fang-Ping

2013-01-01

Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway

(-)-Epicatechin-induced relaxation of isolated human saphenous vein: Roles of K+ and Ca2+ channels.

Science.gov (United States)

Marinko, Marija; Jankovic, Goran; Nenezic, Dragoslav; Milojevic, Predrag; Stojanovic, Ivan; Kanjuh, Vladimir; Novakovic, Aleksandra

2018-02-01

In this study, we aimed to investigate relaxant effect of flavanol (-)-epicatechin on the isolated human saphenous vein (HSV), as a part of its cardioprotective action, and to define the mechanisms underlying this vasorelaxation. (-)-Epicatechin induced a concentration-dependent relaxation of HSV pre-contracted by phenylephrine. Among K + channel blockers, 4-aminopyridine, margatoxin, and iberiotoxin significantly inhibited relaxation of HSV, while glibenclamide considerably reduced effects of the high concentrations of (-)-epicatechin. Additionally, (-)-epicatechin relaxed contraction induced by 80 mM K + , whereas in the presence of nifedipine produced partial relaxation of HSV rings pre-contracted by phenylephrine. In Ca 2+ -free solution, (-)-epicatechin relaxed contraction induced by phenylephrine, but had no effect on contraction induced by caffeine. A sarcoplasmic reticulum Ca 2+ -ATPase inhibitor, thapsigargin, significantly reduced relaxation of HSV produced by (-)-epicatechin. These results demonstrate that (-)-epicatechin produces endothelium-independent relaxation of isolated HSV rings. Vasorelaxation to (-)-epicatechin probably involves activation of 4-aminopyridine- and margatoxin-sensitive K V channels, BK Ca channels, and at least partly, K ATP channels. In addition, not only the inhibition of extracellular Ca 2+ influx, but regulation of the intracellular Ca 2+ release, via inositol-trisphosphate receptors and reuptake into sarcoplasmic reticulum, via stimulation of Ca 2+ -ATPase, as well, most likely participate in (-)-epicatechin-induced relaxation of HSV. Copyright © 2017 John Wiley & Sons, Ltd.

The endoplasmic reticulum stress response in disease ...

African Journals Online (AJOL)

Rafael Vincent M. Manalo

2017-07-12

Jul 12, 2017 ... Review. The endoplasmic reticulum stress response in disease pathogenesis and pathophysiology .... This is an open access article under the CC BY-NC-ND license ... chain binding protein (BIP); however, ER stress permits the release, .... drugs designed to alleviate it often cause more harm long-term.

Regulation of endoplasmic reticulum turnover by selective autophagy

NARCIS (Netherlands)

Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

2015-01-01

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and

Regulation of endoplasmic reticulum turnover by selective autophagy

NARCIS (Netherlands)

Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

2015-01-01

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

Science.gov (United States)

Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

2008-11-01

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Direct measurement of newly synthesized ATP dissociation kinetics in sarcoplasmic reticulum ATPase

International Nuclear Information System (INIS)

Teruel-Puche, J.; Kurzmack, M.; Inesi, G.

1987-01-01

Incubation of SR vesicles with Ca 2+ and ( 32 P)acetylphosphate, yields steady state levels of ( 32 P)phosphorylated enzyme (ATPase) intermediate and high concentrations of Ca 2+ in the lumen of the vesicles. At this time, addition of ADP (and EGTA to lower the Ca 2+ concentration in the medium outside the vesicles) results in single cycle formation of (γ- 32 P)ATP by transfer of ( 32 P)phosphate from the enzyme intermediate to ADP. The phosphoenzyme decay and ATP formation exhibit a fast component within the first 20 msec following addition of ADP, and a slower component reaching an asymptote in approximately 100 msec. They have now measured by a rapid filtration method the fraction of newly synthesized ATP which is bound to the enzyme, as opposed to the fraction dissociated into the medium. They find that nearly all the ATP formed during the initial burst is still bound to the enzyme within the initial 20 msec of reaction. Dissociation of newly synthesized ATP occurs then with approximately 13 sec -1 rate constant, permitting reequilibration of the system and further formation of ATP. The rate limiting effect of ATP dissociation and other partial reactions on the slow component of single cycle ATP synthesis is evaluated by appropriate kinetic simulations

Calcium and Mitosis

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Hepler, P.

1983-01-01

Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

Evidence that the effects of phospholipids on the activity of the Ca(2+)-ATPase do not involve aggregation.

OpenAIRE

Starling, A P; East, J M; Lee, A G

1995-01-01

The Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric from in C12E8, has been reconstituted by dialysis into sealed vesicles of dioleoyl phosphatidylcholine [di(C18:1)PC], dimyristoleoyl phosphatidylcholine [di(C14:1)PC], dinervonyl phosphatidylcholine [di(C24:1)PC] or dipalmitoyl phosphatidylcholine [di(C16:0)PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10,000: 1. Cross-linking experiments show that ATPase molecules are present in these recons...

A turn-on fluorescent probe for endogenous formaldehyde in the endoplasmic reticulum of living cells

Science.gov (United States)

Tang, Yonghe; Ma, Yanyan; Xu, An; Xu, Gaoping; Lin, Weiying

2017-06-01

As the simplest aldehyde compounds, formaldehyde (FA) is implicated in nervous system diseases and cancer. Endoplasmic reticulum is an organelle that plays important functions in living cells. Accordingly, the development of efficient methods for FA detection in the endoplasmic reticulum (ER) is of great biomedical importance. In this work, we developed the first ER-targeted fluorescent FA probe Na-FA-ER. The detection is based on the condensation reaction of the hydrazine group and FA to suppress the photo-induced electron transfer (PET) pathway, resulting in a fluorescence increase. The novel Na-FA-ER showed high sensitivity to FA. In addition, the Na-FA-ER enabled the bio-imaging of exogenous and endogenous FA in living HeLa cells. Most significantly, the new Na-FA-ER was employed to visualize the endogenous FA in the ER in living cells for the first time.

Tributyltin-induced endoplasmic reticulum stress and its Ca2+-mediated mechanism

International Nuclear Information System (INIS)

Isomura, Midori; Kotake, Yaichiro; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-01-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca 2+ signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca 2+ homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca 2+ depletion, and to test this idea, we examined the effect of TBT on intracellular Ca 2+ concentration using fura-2 AM, a Ca 2+ fluorescent probe. TBT increased intracellular Ca 2+ concentration in a TBT-concentration-dependent manner, and Ca 2+ increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca 2+ concentration by releasing Ca 2+ from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca 2+ release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca 2+

Calcium exchange, structure, and function in cultured adult myocardial cells

International Nuclear Information System (INIS)

Langer, G.A.; Frank, J.S.; Rich, T.L.; Orner, F.B.

1987-01-01

Cells digested from adult rat heart and cultured for 14 days demonstrate all the structural elements, in mature form, associated with the process of excitation-contraction (EC) coupling. The transverse tubular (TT) system is well developed with an extensive junctional sarcoplasmic reticulum (JSR). In nonphosphate-containing buffer contraction of the cells is lost as rapidly as zero extracellular Ca concentration ([Ca] 0 ) solution is applied and a negative contraction staircase is produced on increase of stimulation frequency. Structurally and functionally the cells have the characteristics of adult cells in situ. 45 Ca exchange and total 45 Ca measurement in N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES)-buffered perfusate define three components of cellular Ca: 1) a rapidly exchangeable component accounting for 36% of total Ca, 2) a slowly exchangeable component (t/sub 1/2/ 53 min) accounting for 7% total Ca, and 3) the remaining 57% cellular Ca is inexchangeable (demonstrates no significant exchange within 60 min). The slowly exchangeable component can be increased 10-fold within 60 min by addition of phosphate to the perfusate. The Ca distribution and exchange characteristics are little different from those of 3-day cultures of neonatal rat heart previously studied. The results suggest that the cells are representative of adult cells in situ and that both sarcolemmal-bound and sarcoplasmic reticular Ca contribute to the component of Ca that is rapidly exchangeable

Preservation of the positive lusitropic effect of beta-adrenoceptors stimulation in diabetic cardiomyopathy.

Science.gov (United States)

Amour, Julien; Loyer, Xavier; Michelet, Pierre; Birenbaum, Aurélie; Riou, Bruno; Heymes, Christophe

2008-10-01

In diabetic cardiomyopathy, diastolic dysfunction results in part from sarcoplasmic reticulum abnormalities affecting both phospholamban and sarcoplasmic reticulum Ca2+ uptake (SERCA2a). Consequently, the positive lusitropic effect of beta-adrenoceptors stimulation could be altered, and beta3-adrenoceptor over-expression may play a role, as previously demonstrated with an altered positive inotropic effect. In this study, we tested the hypothesis that the beta-adrenergic positive lusitropic effect is altered in diabetic cardiomyopathy, and that beta3-adrenoceptor over-expression is involved. beta-adrenergic responses were investigated in vivo (dobutamine-echocardiography) and in vitro (papillary muscle preparation) in healthy and diabetic rats killed 4 (4W) and 12 (12W) wk after IV streptozotocin injection. The effect of beta3-adrenoceptor pathway inhibition by S-cyanopindolol (selective beta3-adrenoceptor antagonist) or by NG-nitro-L-arginine-methyl-ester (nonselective nitric oxide synthase inhibitor) on the lusitropic response to isoproterenol (nonselective beta-adrenoceptors agonist) was studied in vitro. Western blots were performed to quantify the protein expressions of beta1- and beta3-adrenoceptors, phospholamban, and SERCA2a. Data are presented as mean percentages of baseline+/-sd. Despite the increased phospholamban/SERCA2a protein ratio and documented diastolic dysfunction, the positive lusitropic effect of beta-adrenoceptors stimulation was preserved in vivo (dobutamine) and in vitro (isoproterenol) in 4W and 12W diabetic, compared with healthy, rats. The beta3-adrenoceptor was up-regulated whereas beta1-adrenoceptor was down-regulated in 4W and 12W diabetic, compared with healthy, rats. Nevertheless, S-cyanopindolol or NG-nitro-L-arginine-methyl-ester had no lusitropic effect. The positive lusitropic effect of beta-adrenoceptor stimulation was preserved in diabetic cardiomyopathy. beta3-adrenoceptor over-expression does not seem to affect this process.

Characterization of the functional and anatomical differences in the atrial and ventricular myocardium from three species of elasmobranch fishes

DEFF Research Database (Denmark)

Larsen, Julie; Bushnell, Peter; Steffensen, John

2017-01-01

We assessed the functional properties in atrial and ventricular myocardium (using isolated cardiac strips) of smooth dogfish (Mustelus canis), clearnose skate (Raja eglanteria), and sandbar shark (Carcharhinus plumbeus) by blocking Ca2+ release from the sarcoplasmic reticulum (SR) with ryanodine...... positive first derivative (i.e., contractility), and increased time to 50 % relaxation in atrial tissue from smooth dogfish at 30 °C. It also increased times to peak force and half relaxation in clearnose skate atrial and ventricular tissue at both temperatures, but only in atrial tissue from sandbar shark...... at 30 °C; indicating that SR involvement in excitation–contraction (EC) coupling is species- and temperature-specific in elasmobranch fishes, as it is in teleost fishes. Atrial and ventricular myocardium from all three species displayed a negative force–frequency relationship, but there was no evidence...

Tributyltin-induced endoplasmic reticulum stress and its Ca{sup 2+}-mediated mechanism

Energy Technology Data Exchange (ETDEWEB)

Isomura, Midori; Kotake, Yaichiro, E-mail: yaichiro@hiroshima-u.ac.jp; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-10-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca{sup 2+} signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca{sup 2+} homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca{sup 2+} depletion, and to test this idea, we examined the effect of TBT on intracellular Ca{sup 2+} concentration using fura-2 AM, a Ca{sup 2+} fluorescent probe. TBT increased intracellular Ca{sup 2+} concentration in a TBT-concentration-dependent manner, and Ca{sup 2+} increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca{sup 2+} concentration by releasing Ca{sup 2+} from ER, thereby causing ER stress. - Highlights: • We established that tributyltin induces endoplasmic reticulum (ER) stress. • Tributyltin induces ER stress markers in a concentration-dependent manner. • Tributyltin increases Ca{sup 2+} release from ER, thereby causing ER stress. • Dibutyltin and monobutyltin did not increase GRP78 or intracellular Ca{sup 2+}.

Endoplasmic reticulum stress and diabetic retinopathy

Directory of Open Access Journals (Sweden)

Toshiyuki Oshitari

2008-02-01

Full Text Available Toshiyuki Oshitari1,2, Natsuyo Hata1, Shuichi Yamamoto11Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba City, Chiba, Japan; 2Department of Ophthalmology, Kimitsu Central Hospital, Kisarazu City, Chiba, JapanAbstract: Endoplasmic reticulum (ER stress is involved in the pathogenesis of several diseases including Alzheimer disease and Parkinson disease. Many recent studies have shown that ER stress is related to the pathogenesis of diabetes mellitus, and with the death of pancreatic β-cells, insulin resistance, and the death of the vascular cells in the retina. Diabetic retinopathy is a major complication of diabetes and results in death of both neural and vascular cells. Because the death of the neurons directly affects visual function, the precise mechanism causing the death of neurons in early diabetic retinopathy must be determined. The ideal therapy for preventing the onset and the progression of diabetic retinopathy would be to treat the factors involved with both the vascular and neuronal abnormalities in diabetic retinopathy. In this review, we present evidence that ER stress is involved in the death of both retinal neurons and vascular cells in diabetic eyes, and thus reducing or blocking ER stress may be a potential therapy for preventing the onset and the progression of diabetic retinopathy.Keywords: endoplasmic reticulum stress, diabetic retinopathy, vascular cell death, neuronal cell death

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

International Nuclear Information System (INIS)

Grondin, Melanie; Marion, Michel; Denizeau, Francine; Averill-Bates, Diana A.

2007-01-01

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl - /HCO 3 - exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes

Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

DEFF Research Database (Denmark)

Hansen, Pernille B. Lærkegaard; Friis, Ulla Glenert; Uhrenholt, Torben Rene

2007-01-01

of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP(3), is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 microM) blocked the adenosine-induced constriction whereas...... was abolished by IAA-94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 microM nifedipine (control 8.3 +/- 0.2 microM, ado 3.6 +/- 0.6 microM, ado + nifedipine 6.8 +/- 0.2 microM) suggesting involvement of voltage-dependent calcium channels. CONCLUSION: We conclude...

Localization and in-vivo characterization of thapsia garganica CYP76AE2 indicates a role in thapsigargin biosynthesis

DEFF Research Database (Denmark)

Andersen, Trine Bundgaard; Martinez-Swatson, Karen Agatha; Rasmussen, Silas Anselm

2017-01-01

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+ -ATPase calcium pump in mammals and is of industrial importance as the active...... in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts...

Relaxant mechanisms of 3, 5, 7, 30, 40-pentamethoxyflavone on isolated human cavernosum

DEFF Research Database (Denmark)

Jansakul, Chaweewan; Tachanaparuksa, Kuldej; Mulvany, Michael J.

2012-01-01

We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 30, 40-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted...... Krebs solution with nifedipine (blocker of L-type Ca2þ channels), or in Ca2þ-free Krebs solution, PMF caused a further inhibition of human cavernosum contracted with phenylephrine. In human cavernosum treated with thapsigargin (inhibitor of sarcoplasmic reticulum Ca2þ-ATPase) in Ca2þ-free medium, PMF...

ILDR2: an endoplasmic reticulum resident molecule mediating hepatic lipid homeostasis.

Directory of Open Access Journals (Sweden)

Kazuhisa Watanabe

Full Text Available Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time

Endoplasmic reticulum proteostasis impairment in aging.

Science.gov (United States)

Martínez, Gabriela; Duran-Aniotz, Claudia; Cabral-Miranda, Felipe; Vivar, Juan P; Hetz, Claudio

2017-08-01

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one-third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Ebselen alters cellular oxidative status and induces endoplasmic reticulum stress in rat hippocampal astrocytes.

Science.gov (United States)

Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; de la Casa-Resino, Irene; Martinez-Ruiz, Antonio; Perez-Lopez, Marcos; Portilla, Juan C; Salido, Gines M; Gonzalez, Antonio

2016-05-16

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tannic Acid Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Prostate Cancer.

Science.gov (United States)

Nagesh, Prashanth K B; Hatami, Elham; Chowdhury, Pallabita; Kashyap, Vivek K; Khan, Sheema; Hafeez, Bilal B; Chauhan, Subhash C; Jaggi, Meena; Yallapu, Murali M

2018-03-07

Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G₁ population, due to increase in p18 INK4C and p21 WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

Endoplasmic Reticulum Stress-Mediated Hippocampal Neuron Apoptosis Involved in Diabetic Cognitive Impairment

Directory of Open Access Journals (Sweden)

Xiaoming Zhang

2013-01-01

Full Text Available Poor management of DM causes cognitive impairment while the mechanism is still unconfirmed. The aim of the present study was to investigate the activation of C/EBP Homology Protein (CHOP, the prominent mediator of the endoplasmic reticulum (ER stress-induced apoptosis under hyperglycemia. We employed streptozotocin- (STZ- induced diabetic rats to explore the ability of learning and memory by the Morris water maze test. The ultrastructure of hippocampus in diabetic rats and cultured neurons in high glucose medium were observed by transmission electron microscopy and scanning electron microscopy. TUNEL staining was also performed to assess apoptotic cells while the expression of CHOP was assayed by immunohistochemistry and Western blot assay in these hippocampal neurons. Six weeks after diabetes induction, the escape latency increased and the average frequency in finding the platform decreased in diabetic rats (P<0.05. The morphology of neuron and synaptic structure was impaired; the number of TUNEL-positive cells and the expression of CHOP in hippocampus of diabetic rats and high glucose medium cultured neurons were markedly altered (P<0.05. The present results suggested that the CHOP-dependent endoplasmic reticulum (ER stress-mediated apoptosis may be involved in hyperglycemia-induced hippocampal synapses and neurons impairment and promote the diabetic cognitive impairment.

Plasma membrane—endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis

NARCIS (Netherlands)

Tavassoli, S.; Chao, J.T.; Young, B.P.; Cox, R.C.; Prinz, W.A.; de Kroon, A.I.P.M.; Loewen, C.I.R.

2013-01-01

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and

Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

Energy Technology Data Exchange (ETDEWEB)

Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

2013-06-15

Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes

OpenAIRE

Lavieu, Grégory; Orci, Lelio; Shi, Lei; Geiling, Michael; Ravazzola, Mariella; Wieland, Felix; Cosson, Pierre; Rothman, James E.

2010-01-01

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dime...

Enhanced basal contractility but reduced excitation-contraction coupling efficiency and beta-adrenergic reserve of hearts with increased Cav1.2 activity.

Science.gov (United States)

Tang, Mingxin; Zhang, Xiaoying; Li, Yingxin; Guan, Yinzheng; Ai, Xiaojie; Szeto, Christopher; Nakayama, Hiroyuki; Zhang, Hongyu; Ge, Shuping; Molkentin, Jeffery D; Houser, Steven R; Chen, Xiongwen

2010-08-01

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca(2+) channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the beta-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavbeta2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon beta-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca(2+) current (I(Ca,L)) density, myocyte fractional shortening (FS), peak Ca(2+) transients, and sarcoplasmic reticulum (SR) Ca(2+) content (caffeine-induced Ca(2+) transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the beta-adrenergic agonist isoproterenol (2 microg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of beta-adrenergic stimulation on contraction, Ca(2+) transients, I(Ca,L), and SR Ca(2+) content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.

Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest

International Nuclear Information System (INIS)

Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge

2015-01-01

Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca 2+ , loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest

The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

Energy Technology Data Exchange (ETDEWEB)

Naranmandura, Hua, E-mail: narenman@zju.edu.cn [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Xu, Shi [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Koike, Shota [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Pan, Li Qiang [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Bin [Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Wang, Yan Wei; Rehman, Kanwal; Wu, Bin [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Zhe [Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou (China); Suzuki, Noriyuki, E-mail: n-suzuki@p.chiba-u.ac.jp [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan)

2012-05-01

The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

Endoplasmic reticulum stress causes EBV lytic replication

OpenAIRE

Taylor, Gwen Marie; Raghuwanshi, Sandeep K.; Rowe, David T.; Wadowsky, Robert M.; Rosendorff, Adam

2011-01-01

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)...

Analysis of endoplasmic reticulum of tobacco cells using confocal microscopy

Czech Academy of Sciences Publication Activity Database

Radochová, Barbora; Janáček, Jiří; Schwarzerová, K.; Demjénová, E.; Tomori, Z.; Karen, Petr; Kubínová, Lucie

2005-01-01

Roč. 24, č. 11 (2005), s. 181-185 ISSN 1580-3139 R&D Projects: GA AV ČR(CZ) KJB6011309 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal microscopy * endoplasmic reticulum * image analysis Subject RIV: EA - Cell Biology

4-Phenylbutyrate Benefits Traumatic Hemorrhagic Shock in Rats by Attenuating Oxidative Stress, Not by Attenuating Endoplasmic Reticulum Stress.

Science.gov (United States)

Yang, Guangming; Peng, Xiaoyong; Hu, Yi; Lan, Dan; Wu, Yue; Li, Tao; Liu, Liangming

2016-07-01

Vascular dysfunction such as vascular hyporeactivity following severe trauma and shock is a major cause of death in injured patients. Oxidative stress and endoplasmic reticulum stress play an important role in vascular dysfunction. The objective of the present study was to determine whether or not 4-phenylbutyrate can improve vascular dysfunction and elicit antishock effects by inhibiting oxidative and endoplasmic reticulum stress. Prospective, randomized, controlled laboratory experiment. State key laboratory of trauma, burns, and combined injury. Five hundred and fifty-two Sprague-Dawley rats. Rats were anesthetized, and a model of traumatic hemorrhagic shock was established by left femur fracture and hemorrhage. The effects of 4-phenylbutyrate (5, 20, 50, 100, 200, and 300 mg/kg) on vascular reactivity, animal survival, hemodynamics, and vital organ function in traumatic hemorrhagic shock rats and cultured vascular smooth muscle cells, and the relationship to oxidative stress and endoplasmic reticulum stress was observed. Lower doses of 4-phenylbutyrate significantly improved the vascular function, stabilized the hemodynamics, and increased the tissue blood flow and vital organ function in traumatic hemorrhagic shock rats, and markedly improved the survival outcomes. Among all dosages observed in the present study, 20 mg/kg of 4-phenylbutyrate had the best effect. Further results indicated that 4-phenylbutyrate significantly inhibited the oxidative stress, decreased shock-induced oxidative stress index such as the production of reactive oxygen species, increased the antioxidant enzyme levels such as superoxide dismutase, catalase, and glutathione, and improved the mitochondrial function by inhibiting the opening of the mitochondrial permeability transition pore in rat artery and vascular smooth muscle cells. In contrast, 4-phenylbutyrate did not affect the changes of endoplasmic reticulum stress markers following traumatic hemorrhagic shock. Furthermore, 4

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

Science.gov (United States)

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Acrolein Induces Endoplasmic Reticulum Stress and Causes Airspace Enlargement

Science.gov (United States)

Hanaoka, Masayuki; Natarajan, Ramesh; Kraskauskas, Donatas; Voelkel, Norbert F.

2012-01-01

Background Given the relative abundance and toxic potential of acrolein in inhaled cigarette smoke, it is surprising how little is known about the pulmonary and systemic effects of acrolein. Here we test the hypothesis whether systemic administration of acrolein could cause endoplasmic reticulum (ER) stress, and lung cell apoptosis, leading to the enlargement of the alveolar air spaces in rats. Methods Acute and chronic effects of intraperitoneally administered acrolein were tested. Mean alveolar airspace area was measured by using light microscopy and imaging system software. TUNEL staining and immunohistochemistry (IHC) for active caspase 3 and Western blot analysis for active caspase 3, and caspase 12 were performed to detect apoptosis. The ER-stress related gene expression in the lungs was determined by Quantitative real-time PCR analysis. Acrolein-protein adducts in the lung tissue were detected by IHC. Results Acute administration of acrolein caused a significant elevation of activated caspase 3, upregulation of VEGF expression and induced ER stress proteins in the lung tissue. The chronic administration of acrolein in rats led to emphysematous lung tissue remodeling. TUNEL staining and IHC for cleaved caspase 3 showed a large number of apoptotic septal cells in the acrolein-treated rat lungs. Chronic acrolein administration cause the endoplasmic reticulum stress response manifested by significant upregulation of ATF4, CHOP and GADd34 expression. In smokers with COPD there was a considerable accumulation of acrolein-protein adducts in the inflammatory, airway and vascular cells. Conclusions Systemic administration of acrolein causes endoplasmic reticulum stress response, lung cell apoptosis, and chronic administration leads to the enlargement of the alveolar air spaces and emphysema in rats. The substantial accumulation of acrolein-protein adducts in the lungs of COPD patients suggest a role of acrolein in the pathogenesis of emphysema. PMID:22675432

On The gamma-ray emission from Reticulum II and other dwarf galaxies

Energy Technology Data Exchange (ETDEWEB)

Hooper, Dan; Linden, Tim

2015-09-01

The recent discovery of ten new dwarf galaxy candidates by the Dark Energy Survey (DES) and the Panoramic Survey Telescope and Rapid Response System (Pan-STARRS) could increase the Fermi Gamma-Ray Space Telescope's sensitivity to annihilating dark matter particles, potentially enabling a definitive test of the dark matter interpretation of the long-standing Galactic Center gamma-ray excess. In this paper, we compare the previous analyses of Fermi data from the directions of the new dwarf candidates (including the relatively nearby Reticulum II) and perform our own analysis, with the goal of establishing the statistical significance of any gamma-ray signal from these sources. We confirm the presence of an excess from Reticulum II, with a spectral shape that is compatible with the Galactic Center signal. The significance of this emission is greater than that observed from 99.84% of randomly chosen high-latitude blank-sky locations, corresponding to a local detection significance of 3.2σ. We caution that any dark matter interpretation of this excess must be validated through observations of additional dwarf spheroidal galaxies, and improved calculations of the relative J-factor of dwarf spheroidal galaxies. We improve upon the standard blank-sky calibration approach through the use of multi-wavelength catalogs, which allow us to avoid regions that are likely to contain unresolved gamma-ray sources.

Endoplasmic reticulum redox state is not perturbed by pharmacological or pathological endoplasmic reticulum stress in live pancreatic β-cells.

Directory of Open Access Journals (Sweden)

Irmgard Schuiki

Full Text Available Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor and mCherry protein driven by a GRP78 promoter (UPR-sensor. Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.

Maximal voluntary contraction force, SR function and glycogen resynthesis during the first 72 h after a high-level competitive soccer game

DEFF Research Database (Denmark)

Krustrup, Peter; Ørtenblad, Niels; Nielsen, Joachim

2011-01-01

The aim of this study was to examine maximal voluntary knee-extensor contraction force (MVC force), sarcoplasmic reticulum (SR) function and muscle glycogen levels in the days after a high-level soccer game when players ingested an optimised diet. Seven high-level male soccer players had a vastus...... lateralis muscle biopsy and a blood sample collected in a control situation and at 0, 24, 48 and 72 h after a competitive soccer game. MVC force, SR function, muscle glycogen, muscle soreness and plasma myoglobin were measured. MVC force sustained over 1 s was 11 and 10% lower (P ...

Dietary toxins, endoplasmic reticulum (ER) stress and diabetes.

Science.gov (United States)

Hettiarachchi, Kalindi D; Zimmet, Paul Z; Myers, Mark A

2008-05-01

The incidence of Type 1 diabetes has been increasing at a rate too rapid to be due to changes in genetic risk. Instead changes in environmental factors are the likely culprit. The endoplasmic reticulum (ER) plays an important role in the production of newly synthesized proteins and interference with these processes leads to ER stress. The insulin-producing beta cells are particularly prone to ER stress as a result of their heavy engagement in insulin production. Increasing evidence suggests ER stress is central to initiation and progression of Type 1 diabetes. An early environmental exposure, such as toxins and viral infections, can impart a significant physiological load on beta cells to initiate abnormal processing of proinsulin, ER stress and insulin secretory defects. Release of altered proinsulin from the beta cells early in life may trigger autoimmunity in those with genetic susceptibility leading to cytokine-induced nitric oxide production and so exacerbating ER stress in beta cells, ultimately leading to apoptosis of beta cells and diabetes. Here we suggest that ER stress is an inherent cause of beta cell dysfunction and environmental factors, in particular dietary toxins derived from Streptomyces in infected root vegetables, can impart additional stress that aggravates beta cell death and progression to diabetes. Furthermore, we propose that the increasing incidence of Type 1 diabetes may be accounted for by increased dietary exposure to ER-stress-inducing Streptomyces toxins.

Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury

Directory of Open Access Journals (Sweden)

Fengmei Xing

2017-01-01

Full Text Available Objective. This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R injury via glycogen synthase kinase 3β (GSK-3β and the mitochondrial permeability transition pore (mPTP through inhibition of endoplasmic reticulum stress (ERS. Methods and Results. H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3β phosphorylation and GRP94 expression. NECA increased GSK-3β phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3β and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. Conclusion. These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3β inactivation by NECA.

Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

DEFF Research Database (Denmark)

Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

2011-01-01

phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p......Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...

The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

Science.gov (United States)

Bellucci, Michele; De Marchis, Francesca; Pompa, Andrea

2017-12-18

The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Hepatic glycogen synthesis in the fetal mouse: An ultrastructural, morphometric, and autoradiographic investigation of the relationship between the smooth endoplasmic reticulum and glycogen

International Nuclear Information System (INIS)

Breslin, J.S.

1989-01-01

Fetal rodent hepatocytes undergo a rapid and significant accumulation of glycogen prior to birth. The distinct association of the smooth endoplasmic reticulum (SER) with glycogen during glycogen synthesis documented in the adult hepatocyte has not been clearly demonstrated in the fetus. The experiments described in this dissertation tested the hypothesis that SER is present and functions in the synthesis of fetal hepatic glycogen. Biochemical analysis, light microscopic (LM) histochemistry and electron microscope (EM) morphometry demonstrated that fetal hepatic glycogen synthesis began on day 15, with maximum accumulation occurring between days 17-19. Glycogen accumulation began in a small population of cells. Both the number of cells containing glycogen and the quantity of glycogen per cell increased as glycogen accumulated. Smooth endoplasmic reticulum (SER) was observed on day 14 of gestation and throughout fetal hepatic glycogen synthesis, primarily as dilated ribosome-free terminal extensions of rough endoplasmic reticulum (RER), frequently associated with glycogen. SER was in close proximity to isolated particles of glycogen and at the periphery of large compact glycogen deposits. Morphometry demonstrated that the membrane surface of SER in the average fetal hepatocyte increased as glycogen accumulated through day 18 and dropped significantly as glycogen levels peaked on day 19. Parallel alterations in RER membrane surface, indicated overall increases in ER membrane surface. Autoradiography following administration of 3 H-galactose demonstrated that newly synthesized glycogen was deposited near profiles of SER at day 16 and at day 18; however, at day 18 the majority of label was uniformly distributed over glycogen remote from profiles of SER

Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

LENUS (Irish Health Repository)

Semjonous, Nina M

2011-01-01

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

A multiscale active structural model of the arterial wall accounting for smooth muscle dynamics.

Science.gov (United States)

Coccarelli, Alberto; Edwards, David Hughes; Aggarwal, Ankush; Nithiarasu, Perumal; Parthimos, Dimitris

2018-02-01

Arterial wall dynamics arise from the synergy of passive mechano-elastic properties of the vascular tissue and the active contractile behaviour of smooth muscle cells (SMCs) that form the media layer of vessels. We have developed a computational framework that incorporates both these components to account for vascular responses to mechanical and pharmacological stimuli. To validate the proposed framework and demonstrate its potential for testing hypotheses on the pathogenesis of vascular disease, we have employed a number of pharmacological probes that modulate the arterial wall contractile machinery by selectively inhibiting a range of intracellular signalling pathways. Experimental probes used on ring segments from the rabbit central ear artery are: phenylephrine, a selective α 1-adrenergic receptor agonist that induces vasoconstriction; cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase; and ryanodine, a diterpenoid that modulates Ca 2+ release from the sarcoplasmic reticulum. These interventions were able to delineate the role of membrane versus intracellular signalling, previously identified as main factors in smooth muscle contraction and the generation of vessel tone. Each SMC was modelled by a system of nonlinear differential equations that account for intracellular ionic signalling, and in particular Ca 2+ dynamics. Cytosolic Ca 2+ concentrations formed the catalytic input to a cross-bridge kinetics model. Contractile output from these cellular components forms the input to the finite-element model of the arterial rings under isometric conditions that reproduces the experimental conditions. The model does not account for the role of the endothelium, as the nitric oxide production was suppressed by the action of L-NAME, and also due to the absence of shear stress on the arterial ring, as the experimental set-up did not involve flow. Simulations generated by the integrated model closely matched experimental

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

DEFF Research Database (Denmark)

Lotti, L V; Lanfrancone, L; Migliaccio, E

1996-01-01

area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

Left ventricular wall stress and sarcoplasmic reticulum Ca(2+)-ATPase gene expression in renal hypertensive rats: dose-dependent effects of ACE inhibition and AT1-receptor blockade.

Science.gov (United States)

Zierhut, W; Studer, R; Laurent, D; Kästner, S; Allegrini, P; Whitebread, S; Cumin, F; Baum, H P; de Gasparo, M; Drexler, H

1996-05-01

Cardiac hypertrophy is associated with altered Ca2+ handling and may predispose to the development of LV dysfunction and cardiac failure. At the cellular level, the re-expression of ANF represents a well-established marker of myocyte hypertrophy while the decreased expression of the sarcoplasmatic reticulum (SR) Ca(2+)-ATPase is thought o play a crucial role in the alterations of Ca2+ handling and LV function. We assessed the dose-dependent effect of chronic ACE inhibition or AT1 receptor blockade on cardiac function in relation to the cardiac expression of the SR Ca(2+)-ATPase and ANF. Renal hypertensive rats (2K-1C) were treated for 12 weeks with three different doses of the ACE inhibitor benazepril, the AT1-receptor antagonist valsartan (each drug 0.3, 3, and 10 mg/kg per day i.p.) or placebo. LV dimensions, hypertrophy and wall stress were determined in vivo by magnetic resonance imaging and the gene expressions of ANF and SR Ca(2+)-ATPase were quantified by Northern blot. Low doses of both drugs did not affect blood pressure, hypertrophy, systolic wall stress and the ANF and SR Ca(2+)-ATPase gene expression. High doses of each drug reduced systolic blood pressure, wall stress, and LV hypertrophy to a similar extent and to values comparable to normotensive, age-matched rats. In addition, high dose treatment reduced LV end-systolic and end-diastolic volume as compared to untreated 2K-1C animals and normalized the mRNA levels of both ANF and SR Ca(2+)-ATPase (as compared to normotensive animals). We conclude that in this model, high doses of ACE inhibition and AT1-receptor blockade are necessary to normalize systolic blood pressure, LV hypertrophy and systolic LV wall stress which, in turn, is associated with restoration of a normal cardiac phenotype with respect to SR Ca(2+)-ATPase and ANF and normalization of cardiac function.

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

Energy Technology Data Exchange (ETDEWEB)

Mondal, Tapan Kumar; Emeny, Rebecca T.; Gao, Donghong; Ault, Jeffrey G.; Kasten-Jolly, Jane; Lawrence, David A., E-mail: david.lawrence@health.ny.gov

2015-12-01

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2 h, host defenses against bacterial growth are suppressed 2–3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidation (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes. - Highlights: • Cold-restraint (physical/psychological stress) induces early oxidative stress. • The oxidative stress relates to catecholamine signaling beta-adrenoceptors. • Physical/psychological stress combines infection enhancing inflammation. • Endoplasmic reticulum

Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells.

Science.gov (United States)

Knowles, Michelle K; Guenza, Marina G; Capaldi, Roderick A; Marcus, Andrew H

2002-11-12

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10(-2) to 10(3) s and 0.5-2.5 microm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,tau), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

Role of glycogen availability in sarcoplasmic reticulum Ca2+ kinetics in human skeletal muscle

DEFF Research Database (Denmark)

Ørtenblad, Niels; Nielsen, Joachim; Saltin, Bengt

2011-01-01

Glucose is stored as glycogen in skeletal muscle. The importance of glycogen as a fuel during exercise has been recognized since the 1960s; however, little is known about the precise mechanism that relates skeletal muscle glycogen to muscle fatigue. We show that low muscle glycogen is associated...... with an impairment of muscle ability to release Ca(2+), which is an important signal in the muscle activation. Thus, depletion of glycogen during prolonged, exhausting exercise may contribute to muscle fatigue by causing decreased Ca(2+) release inside the muscle. These data provide indications of a signal...

The orphan nuclear receptor NR4A1 (Nur77) regulates oxidative and endoplasmic reticulum stress in pancreatic cancer cells.

Science.gov (United States)

Lee, Syng-Ook; Jin, Un-Ho; Kang, Jeong Han; Kim, Sang Bae; Guthrie, Aaron S; Sreevalsan, Sandeep; Lee, Ju-Seog; Safe, Stephen

2014-04-01

NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNA interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum stress, including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and endoplasmic reticulum stress by these agents was attenuated after cotreatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and endoplasmic reticulum stress; thus, demonstrating that NR4A1 regulates levels of endoplasmic reticulum stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of the ROS/endoplasmic reticulum stress and proapoptotic pathways. The NR4A1 receptor is pro-oncogenic, regulates the ROS/endoplasmic reticulum stress pathways, and inactivation of the receptor represents a novel pathway for inducing cell death in pancreatic cancer. Mol Cancer Res; 12(4); 527-38. ©2014 AACR.

Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

Science.gov (United States)

Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

2016-01-01

Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

Detection of Sequence-Specific Tyrosine Nitration of Manganese SOD and SERCA in Cardiovascular Disease and Aging

Energy Technology Data Exchange (ETDEWEB)

Xu, Shanqin; Ying, Jia; Jiang, Bingbing; Guo, Wei; Adachi, Takeshi; Sharov, Victor; Lazar, Harold; Menzoian, James; Knyushko, Tanya V.; Bigelow, Diana J.; Schoneich, Christian; Cohen, Richard

2006-06-01

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies towards site-specific nY-modified proteins and to use histochemical and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies towards peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with angiotensin II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite that chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site, but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates, but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

Science.gov (United States)

Vinogradova, Tatiana M.; Lakatta, Edward G.

2009-01-01

Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

Structure-function relation of phospholamban: modulation of channel activity as a potential regulator of SERCA activity.

Directory of Open Access Journals (Sweden)

Serena Smeazzetto

Full Text Available Phospholamban (PLN is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca(2+-ATPase (SERCA in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C or destabilize (I47A the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca(2+ accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca(2+ uptake. A reduced total conductance of the K(+ transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca(2+ uptake in the same way as an inhibition of K(+ channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.

Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

Science.gov (United States)

Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

2017-04-01

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

Hypothalamic endoplasmic reticulum stress of overtrained mice after recovery

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Ana P. Pinto

2017-05-01

Full Text Available Abstract AIMS knowing the relationship between endoplasmic reticulum (ER stress and inflammation and based on the fact that downhill running-based overtraining (OT model increases hypothalamus levels of some pro-inflammatory cytokines, we verified the effects of three OT protocols on the levels of BiP, pIRE-1 (Ser734, pPERK (Thr981, pelF2alpha (Ser52, ATF-6 and GRP-94 proteins in the mouse hypothalamus after two weeks of recovery. METHODS the mice were randomized into control (CT, overtrained by downhill running (OTR/down, overtrained by uphill running (OTR/up and overtrained by running without inclination (OTR groups. After 2-week total recovery period (i.e., week 10, hypothalamus was removed and used for immunoblotting. RESULTS the OTR/down group exhibited high levels of BiP and ATF6. The other OT protocols showed higher levels of pPERK (Th981 and pelf-2alpha (Ser52 when compared with the CT group. CONCLUSION the current results suggest that after a 2-week total recovery period, the overtrained groups increased partially their ER stress protein levels, but without hypothalamic inflammation, which characterizes a physiological condition related to an adaptation mechanism.

Ricin A chain reaches the endoplasmic reticulum after endocytosis

International Nuclear Information System (INIS)

Liu Qiong; Zhan Jinbiao; Chen Xinhong; Zheng Shu

2006-01-01

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum

Hypothyroidism Causes Endoplasmic Reticulum Stress in Adult Rat Hippocampus: A Mechanism Associated with Hippocampal Damage

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Alejandra Paola Torres-Manzo

2018-01-01

Full Text Available Thyroid hormones (TH are essential for hippocampal neuronal viability in adulthood, and their deficiency causes hypothyroidism, which is related to oxidative stress events and neuronal damage. Also, it has been hypothesized that hypothyroidism causes a glucose deprivation in the neuron. This study is aimed at evaluating the temporal participation of the endoplasmic reticulum stress (ERE in hippocampal neurons of adult hypothyroid rats and its association with the oxidative stress events. Adult Wistar male rats were divided into euthyroid and hypothyroid groups. Thyroidectomy with parathyroid gland reimplementation caused hypothyroidism at three weeks postsurgery. Oxidative stress, redox environment, and antioxidant enzyme markers, as well as the expression of the ERE through the pathways of PERK, ATF6, and IRE1, were evaluated at the 3rd and 4th weeks postsurgery. We found a rise in ROS and nitrite production; also, catalase increased and glutathione peroxidase diminished their activities. These events promote an enhancement of the lipoperoxidation, as well as of γ-GT, myeloperoxidase, and caspase 3 activities. With respect to ERE, there were ATF6, IRE1, and GADD153 overexpressions with a reduction in mitochondrial activity and GSH2/GSSG ratio. We conclude that the endoplasmic reticulum stress might play a pivotal role in the activation of hypothyroidism-induced hippocampal cell death.

Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress.

Science.gov (United States)

Kanemoto, Soshi; Nitani, Ryota; Murakami, Tatsuhiko; Kaneko, Masayuki; Asada, Rie; Matsuhisa, Koji; Saito, Atsushi; Imaizumi, Kazunori

2016-11-11

The endoplasmic reticulum (ER) plays a pivotal role in maintaining cellular homeostasis. However, numerous environmental and genetic factors give rise to ER stress by inducing an accumulation of unfolded proteins. Under ER stress conditions, cells initiate the unfolded protein response (UPR). Here, we demonstrate a novel aspect of the UPR by electron microscopy and immunostaining analyses, whereby multivesicular body (MVB) formation was enhanced after ER stress. This MVB formation was influenced by inhibition of ER stress transducers inositol required enzyme 1 (IRE1) and PKR-like ER kinase (PERK). Furthermore, exosome release was also increased during ER stress. However, in IRE1 or PERK deficient cells, exosome release was not upregulated, indicating that IRE1- and PERK-mediated pathways are involved in ER stress-dependent exosome release. Copyright © 2016 Elsevier Inc. All rights reserved.

Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Bao, Jichen; Huang, Mingtao; Petranovic, Dina

2017-01-01

in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species...... were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance....

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

Science.gov (United States)

Belemtougri, R.G.; Constantin, B.; Cognard, C.; Raymond, G.; Sawadogo, L.

2006-01-01

Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explaine their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release. PMID:16365927

Ca2+-clock-dependent pacemaking in the sinus node is impaired in mice with a cardiac specific reduction in SERCA2 abundance

Directory of Open Access Journals (Sweden)

Sunil Jit Ramamoorthy Jeewanlal Logantha

2016-06-01

Full Text Available Background: The sarcoplasmic reticulum Ca2+-ATPase (SERCA2 pump is an important component of the Ca2+-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO as a model for investigating SERCA2’s role in sinus node pacemaking.Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P70% Serca2 downregulation.Conclusions: Serca2 KO mice show a disrupted Ca2+-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function.

The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana

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DANIELA C URBINA

2006-01-01

Full Text Available Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 µM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism

The road to understanding an ion pump

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Toyoshima, Chikashi

2016-04-01

In the past 25 years or so I have been working almost exclusively on two proteins: the Ca2+-ATPase of muscle sarcoplasmic reticulum, and the Na+, K+-ATPase expressed in all animal cells, both are membrane ion pumps representing P-type ion translocating ATPases. My ambition as a scientist is to completely understand the meaning of their atomic structures. How I became a scientist is described elsewhere (Nuzzo R 2006 Proc. Natl. Acad. Sci. USA 103 1165-7), and focus here is given to my struggle towards a deep understanding of Ca2+-ATPase. This is a long but very fascinating and rewarding journey.

Organellar Calcium Buffers

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Prins, Daniel; Michalak, Marek

2011-01-01

Ca2+ is an important intracellular messenger affecting many diverse processes. In eukaryotic cells, Ca2+ storage is achieved within specific intracellular organelles, especially the endoplasmic/sarcoplasmic reticulum, in which Ca2+ is buffered by specific proteins known as Ca2+ buffers. Ca2+ buffers are a diverse group of proteins, varying in their affinities and capacities for Ca2+, but they typically also carry out other functions within the cell. The wide range of organelles containing Ca2+ and the evidence supporting cross-talk between these organelles suggest the existence of a dynamic network of organellar Ca2+ signaling, mediated by a variety of organellar Ca2+ buffers. PMID:21421925

Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

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Shengzhang Lin

2013-01-01

Full Text Available Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990 with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153, a marker of the endoplasmic-reticulum-stress- (ERS- mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78, phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK, and phosphoeukaryotic initiation factor-2α (phospho-eIF2α, activating transcription factor 4 (ATF4 and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

International Nuclear Information System (INIS)

Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

2009-01-01

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

NARCIS (Netherlands)

Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D

1998-01-01

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by

Sarco/Endoplasmic reticulum Ca2+-ATPases (SERCA contribute to GPCR-mediated taste perception.

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Naoko Iguchi

Full Text Available The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory, bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca(2+ concentration ([Ca(2+](c for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca(2+](c from the cytosol of taste receptor cells (TRCs and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca(2+ clearance in TRCs, we sought the molecules involved in [Ca(2+](c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA family that sequesters cytosolic Ca(2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca(2+ release for signaling. Serca3-knockout (KO mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca(2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception.

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

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Fujimoto, Michiko [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Hayashi, Teruo, E-mail: thayashi@mail.nih.gov [Cellular Stress Signaling Unit, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States); Su, Tsung-Ping, E-mail: tsu@intra.nida.nih.gov [Cellular Pathobiology Section, Integrative Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD 21224 (United States)

2012-01-06

Highlights: Black-Right-Pointing-Pointer The endoplasmic reticulum subdomain termed MAM associates with mitochondria. Black-Right-Pointing-Pointer The biophysical role of lipids in the MAM-mitochondria association is unknown. Black-Right-Pointing-Pointer The in vitro membrane association assay was used to examine the role of lipids. Black-Right-Pointing-Pointer Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP{sub 3} receptor-mediated Ca{sup 2+} influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-{beta}-cyclodextrin (M{beta}C) significantly increases the association between MAMs and mitochondria, whereas M{beta}C saturated with cholesterol does not change the association. {sup 14}C-Serine pulse-labeling demonstrated that the treatment of living cells with M{beta}C decreases the level of de novo synthesized {sup 14}C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of

The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria

International Nuclear Information System (INIS)

Fujimoto, Michiko; Hayashi, Teruo; Su, Tsung-Ping

2012-01-01

Highlights: ► The endoplasmic reticulum subdomain termed MAM associates with mitochondria. ► The biophysical role of lipids in the MAM–mitochondria association is unknown. ► The in vitro membrane association assay was used to examine the role of lipids. ► Cholesterol was found to negatively regulate the association. -- Abstract: The unique endoplasmic reticulum (ER) subdomain termed the mitochondria-associated ER membrane (MAM) engages the physical connection between the ER and the mitochondrial outer membrane and plays a role in regulating IP 3 receptor-mediated Ca 2+ influx and the phospholipid transport between the two organelles. The MAM contains certain signaling and membrane-tethering proteins but also lipids including cholesterol. The biophysical role of lipids at the MAM, specifically in the physical interaction between the MAM of the ER and mitochondria, remains not totally clarified. Here we employed the in vitro membrane association assay to investigate the role of cholesterol in the association between MAMs and mitochondria. The purified MAMs and mitochondria were mixed in vitro in a test tube and then the physical association of the two subcellular organelles was quantified indirectly by measuring the presence of the MAM-specific protein sigma-1 receptors in the mitochondria fraction. Purified MAMs contained free cholesterol approximately 7 times higher than that in microsomes. We found that depletion of cholesterol in MAMs with methyl-β-cyclodextrin (MβC) significantly increases the association between MAMs and mitochondria, whereas MβC saturated with cholesterol does not change the association. 14 C-Serine pulse-labeling demonstrated that the treatment of living cells with MβC decreases the level of de novo synthesized 14 C-phosphatidylserine (PtSer) and concomitantly increases greatly the synthesis of 14 C-phosphatidylethanolamine (PtEt). Apparently, cholesterol depletion increased the PtSer transport from MAMs to mitochondria. Our

TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

International Nuclear Information System (INIS)

Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing

2014-01-01

Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR 1 pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR 1 , TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR 1 was suppressed with its siRNA. The protein levels of TNFα, TNFR 1 and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR 1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR 1 , Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR 1 –siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR 1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and concentration-dependent manners. �

Endoplasmic reticulum stress-induced resistance to doxorubicin is reversed by paeonol treatment in human hepatocellular carcinoma cells.

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Lulu Fan

Full Text Available BACKGROUND: Endoplasmic reticulum stress (ER stress is generally activated in solid tumors and results in tumor cell anti-apoptosis and drug resistance. Paeonol (Pae, 2-hydroxy-4-methoxyacetophenone, is a natural product extracted from the root of Paeonia Suffruticosa Andrew. Although Pae displays anti-neoplastic activity and increases the efficacy of chemotherapeutic drugs in various cell lines and in animal models, studies related to the effect of Pae on ER stress-induced resistance to chemotherapeutic agents in hepatocellular carcinoma (HCC are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the effect of the endoplasmic reticulum (ER stress response during resistance of human hepatocellular carcinoma cells to doxorubicin. Treatment with the ER stress-inducer tunicamycin (TM before the addition of doxorubicin reduced the rate of apoptosis induced by doxorubicin. Interestingly, co-pretreatment with tunicamycin and Pae significantly increased apoptosis induced by doxorubicin. Furthermore, induction of ER stress resulted in increasing expression of COX-2 concomitant with inactivation of Akt and up-regulation of the pro-apoptotic transcription factor CHOP (GADD153 in HepG2 cells. These cellular changes in gene expression and Akt activation may be an important resistance mechanism against doxorubicin in hepatocellular carcinoma cells undergoing ER stress. However, co-pretreatment with tunicamycin and Pae decreased the expression of COX-2 and levels of activation of Akt as well as increasing the levels of CHOP in HCC cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Pae reverses ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells by targeting COX-2 mediated inactivation of PI3K/AKT/CHOP.

Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

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Jeffrey Thomas Cole

2012-04-01

Full Text Available Brain cells expend large amounts of energy sequestering calcium (Ca2+, while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P, a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA. Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1-10 mM. The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity

Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle

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Koh, Ho-Jin; Toyoda, Taro; Didesch, Michelle M.; Lee, Min-Young; Sleeman, Mark W.; Kulkarni, Rohit N.; Musi, Nicolas; Hirshman, Michael F.; Goodyear, Laurie J.

2013-01-01

Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle. PMID:23695665

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

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A.J. Parodi

1998-05-01

Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

Science.gov (United States)

Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

2018-06-01

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

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Yu-Ping Hsiao

2015-01-01

Full Text Available Malic acid (MA has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT. The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs. Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR was significantly decreased whereas extracellular acidification rate (ECAR was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

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Zhang, Fu-Tao; Ding, Yi; Shah, Zahir; Xing, Dan; Gao, Yuan; Liu, Dong Ming; Ding, Ming-Xing, E-mail: dmx@mail.hzau.edu.cn

2014-04-15

Background and purpose: Quinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR{sub 1} pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approach: Canine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR{sub 1}, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR{sub 1} was suppressed with its siRNA. The protein levels of TNFα, TNFR{sub 1} and caspase-12 were assayed using Western blotting. Key results: The survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR{sub 1} and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR{sub 1}, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR{sub 1}–siRNA interference. Conclusions and implications: Ofloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR{sub 1} pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage. - Highlights: • Chondrocyte apoptosis is induced by ofloxacin in a time- and

Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle.

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Kanzaki, Keita; Watanabe, Daiki; Kuratani, Mai; Yamada, Takashi; Matsunaga, Satoshi; Wada, Masanobu

2017-02-01

The aim of this study was to examine the in vivo effects of eccentric contraction (ECC) on calpain-dependent proteolysis of Ca 2+ -regulatory proteins and force production in fast-twitch skeletal muscles. Rat extensor digitorum longus muscles were exposed to 200 repeated ECC in situ and excised immediately [recovery 0 (REC0)] or 3 days [recovery 3 (REC3)] after cessation of ECC. Calpain inhibitor (CI)-treated rats were intraperitoneally injected with MDL-28170 before ECC and during REC3. Tetanic force was markedly reduced at REC0 and remained reduced at REC3. CI treatment ameliorated the ECC-induced force decline but only at REC3. No evidence was found for proteolysis of dihydropyridine receptor (DHPR), junctophilin (JP)1, JP2, ryanodine receptor (RyR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)1a, or junctional face protein-45 at REC0. At REC3, ECC resulted in decreases in DHPR, JP1, JP2, RyR, and SERCA1a. CI treatment prevented the decreases in DHPR, JP1, and JP2, whereas it had little effect on RyR and SERCA1a. These findings suggest that DHPR, JP1, and JP2, but not RyR and SERCA1a, undergo calpain-dependent proteolysis in in vivo muscles subjected to ECC and that impaired function of DHPR and/or JP might cause prolonged force deficits with ECC. NEW & NOTEWORTHY Calpain-dependent proteolysis is one of the contributing factors to muscle damage that occurs with eccentric contraction (ECC). It is unclear, however, whether calpains account for proteolysis of Ca 2+ -regulatory proteins in in vivo muscles subjected to ECC. Here, we provide evidence that dihydropyridine receptor and junctophilin, but not ryanodine receptor and sarcoplasmic reticulum Ca 2+ -ATPase, undergo calpain-dependent proteolysis. Copyright © 2017 the American Physiological Society.

Development of porcine model of chronic tachycardia-induced cardiomyopathy.

Science.gov (United States)

Paslawska, Urszula; Gajek, Jacek; Kiczak, Liliana; Noszczyk-Nowak, Agnieszka; Skrzypczak, Piotr; Bania, Jacek; Tomaszek, Alicja; Zacharski, Maciej; Sambor, Izabela; Dziegiel, Piotr; Zysko, Dorota; Banasiak, Waldemar; Jankowska, Ewa A; Ponikowski, Piotr

2011-11-17

There are few experimental models of heart failure (HF) in large animals, despite structural and functional similarities to human myocardium. We have developed a porcine model of chronic tachycardia-induced cardiomyopathy. Homogenous siblings of White Large breed swine (n=6) underwent continuous right ventricular (RV) pacing at 170 bpm; 2 subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were presented for euthanasia at subsequent stages: mild, moderate and end-stage HF. Left ventricle (LV) sections were analyzed histologically and relative ANP, BNP, phospholamban and sarcoplasmic reticulum calcium ATPase 2a transcript levels in LV were quantified by real time RT-PCR. In the course of RV pacing, animals demonstrated reduced exercise capacity (time of running until being dyspnoeic: 6.6 ± 0.5 vs. 2.4 ± 1.4 min), LV dilatation (LVEDD: 4.9 ± 0.4 vs. 6.7 ± 0.4 cm), impaired LV systolic function (LVEF: 69 ± 8 vs. 32 ± 7 %), (all baseline vs. before euthanasia, all p<0.001). LV tissues from animals with moderate and end-stage HF demonstrated local foci of interstitial fibrosis, congestion, cardiomyocyte hypertrophy and atrophy, which was not detected in controls and mild HF animals. The up-regulation of ANP and BNP and a reduction in a ratio of sarcoplasmic reticulum calcium ATPase 2a and phospholamban in failing myocardium were observed as compared to controls. In pigs, chronic RV pacing at relatively low rate can be used as an experimental model of HF, as it results in a gradual deterioration of exercise tolerance accompanied by myocardial remodeling confirmed at subcellular level. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

THE RESPONSE OF DISSEMINATED RETICULUM CELL SARCOMA TO THE INTRAVENOUS INJECTION OF COLLOIDAL RADIOACTIVE GOLD

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Rubin, Philip; Levitt, Seymour H.

1963-06-15

Case histories of two patients treated with colloidal radiogold for diffuse reticulum cell sarcoma are presented. Further analysis of the method is suggested by the unusually long survival time of one of the patients. It was concluded that, although external radiotherapy remains the treatment of choice in localized reticulum cell sarcoma, intravenous colloidal radiogold may be a useful agent in lymphosarcomas with diffuse minute neoplastic liver and spleen involvements. Intravenous colloidal radiogold can produce bone marrow depression and thrombocytopenia which can lead to death. This factor tends to argue against therapeutic use of the agent. It is suggested that no more than 50 mC Au/sup 198/ intravenously should be used for treatment of this disease. (R.M.G.)

Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

LENUS (Irish Health Repository)

Samali, Afshin

2010-01-01

The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response

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Afshin Samali

2010-01-01

Full Text Available The endoplasmic reticulum (ER is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR. The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

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Saheki, Yasunori; De Camilli, Pietro

2017-06-20

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

DEFF Research Database (Denmark)

Mahmmoud, Yasser Ahmed

2005-01-01

Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others...... have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase. We tested the hypothesis that curcumin may modulate other members of the P2-type ATPase superfamily by studying the effects of curcumin on the activity...... and kinetic properties of the Na,K-ATPase. Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K0.514.6 M). Curcumin decreased the apparent affinity of Na,K-ATPase for K+ and increased it for Na+ and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction...

Increased classical endoplasmic reticulum stress is sufficient to reduce chondrocyte proliferation rate in the growth plate and decrease bone growth.

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Louise H W Kung

Full Text Available Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED and pseudoachondroplasia (PSACH. The majority of these diseases feature classical endoplasmic reticulum (ER stress and activation of the unfolded protein response (UPR as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2α and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole.

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

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Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

2013-03-01

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Lipid Droplet Formation Is Dispensable for Endoplasmic Reticulum-associated Degradation*

OpenAIRE

Olzmann, James A.; Kopito, Ron R.

2011-01-01

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an “escape hatch” through which misfolded proteins, toxins, and viruses can exit ...

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

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Bravo, Roberto; Gutierrez, Tomás; Paredes, Felipe; Gatica, Damián; Rodriguez, Andrea E.; Pedrozo, Zully; Chiong, Mario; Parra, Valentina; Quest, Andrew F.G.; Rothermel, Beverly A.; Lavandero, Sergio

2014-01-01

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER–mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders. PMID:22064245

Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

Energy Technology Data Exchange (ETDEWEB)

Feng, Shan-Li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Sun, Ming-Rui [Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001 (China); Li, Ting-Ting; Yin, Xin [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Xu, Chang-Qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Sun, Yi-Hua, E-mail: syh200415@126.com [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China)

2011-03-11

Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

CCPG1, a cargo receptor required for reticulophagy and endoplasmic reticulum proteostasis.

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Smith, Matthew D; Wilkinson, Simon

2018-06-19

The importance of selective macroautophagy/autophagy in cellular health is increasingly evident. The selective degradation of portions of the endoplasmic reticulum (ER), or reticulophagy, is an emerging example but requires further mechanistic detail and broad evidence of physiological relevance. In a recent study, we identified CCPG1, an ER-resident transmembrane protein that can bind to Atg8-family proteins and, independently and discretely, to RB1CC1/FIP200. Both of these interactions are required to facilitate CCPG1's function as a reticulophagy cargo receptor. CCPG1 transcripts are inducible by ER stress, providing a direct link between ER stress and reticulophagy. In vivo, CCPG1 prevents the hyper-accumulation of insoluble protein within the ER lumen of pancreatic acinar cells and alleviates ER stress. Accordingly, CCPG1 loss sensitizes the exocrine pancreas to tissue injury.

Variable Stars in Large Magellanic Cloud Globular Clusters. III. Reticulum

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Kuehn, Charles A.; Dame, Kyra; Smith, Horace A.; Catelan, Márcio; Jeon, Young-Beom; Nemec, James M.; Walker, Alistair R.; Kunder, Andrea; Pritzl, Barton J.; De Lee, Nathan; Borissova, Jura

2013-06-01

This is the third in a series of papers studying the variable stars in old globular clusters in the Large Magellanic Cloud. The primary goal of this series is to look at how the characteristics and behavior of RR Lyrae stars in Oosterhoff-intermediate systems compare to those of their counterparts in Oosterhoff-I/II systems. In this paper we present the results of our new time-series BVI photometric study of the globular cluster Reticulum. We found a total of 32 variables stars (22 RRab, 4 RRc, and 6 RRd stars) in our field of view. We present photometric parameters and light curves for these stars. We also present physical properties, derived from Fourier analysis of light curves, for some of the RR Lyrae stars. We discuss the Oosterhoff classification of Reticulum and use our results to re-derive the distance modulus and age of the cluster. Based on observations taken with the SMARTS 1.3 m telescope operated by the SMARTS Consortium and observations taken at the Southern Astrophysical Research (SOAR) telescope, which is a joint project of the Ministério da Ciência, Tecnologia, e Inovação (MCTI) da República Federativa do Brasil, the U.S. National Optical Astronomy Observatory (NOAO), the University of North Carolina at Chapel Hill (UNC), and Michigan State University (MSU).

Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

OpenAIRE

Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

2011-01-01

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit t...

Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells

DEFF Research Database (Denmark)

Raciti, G A; Iadicicco, C; Ulianich, L

2010-01-01

Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells.......Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells....

Single-prolonged stress induces endoplasmic reticulum-dependent apoptosis in the hippocampus in a rat model of post-traumatic stress disorder.

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Fang Han

Full Text Available BACKGROUND: Our previous research indicated that apoptosis induced atrophy in the hippocampus of post-traumatic stress disorder (PTSD rats. Endoplasmic reticulum (ER stress-induced apoptosis has been implicated in the development of several disorder diseases. The aim of this study was to investigate whether endoplasmic reticulum-related pathway is involved in single-prolonged stress (SPS induces apoptosis in the hippocampus of PTSD rats by examining the expression levels of three important indicators in the ER-related apoptotic pathway: Glucose-regulated protein (GRP 78, caspase-12 and Ca(2+/CaM/CaMkinaseIIα (CaMkIIα. METHODS: Wistar rats were sacrificed at 1, 4 and 7 days after SPS. SPS is a reliable animal model of PTSD. The apoptotic cells in the hippocampus were assessed by TUNEL method and transmission electron microscopy (TEM. Free intracellular Ca(2+ concentration was measured. GRP78 expression was examined by immunohistochemistry, western blotting and RT-PCR. mRNA of caspase-12 and CaM/CaMkIIα were determined by RT-PCR. RESULTS: Our results showed that apoptotic cells were increased in the SPS rats. TEM analysis revealed characteristic morphological changes of apoptosis in these cells. We observed that GRP78 was significantly up-regulated during early PTSD, and then recovered at 7 days after SPS. By RT-PCR, we observed that the change in caspase-12 expression level was similar to that in GRP78. Moreover, the free intracellular Ca(2+ concentration was significantly higher at 1 day after SPS and decreased in 7 days. CaM expression increased significantly, while CaMKIIα expression decreased significantly in the hippocampus at 1 day after SPS. CONCLUSION: SPS induced change in the expression levels of GRP78, caspase-12 and Ca(2+/CaM/CaMkIIα in the hippocampus of PTSD rats indicated that the endoplasmic reticulum pathway may be involved in PTSD-induced apoptosis.

Role of endoplasmic reticulum stress in acrolein-induced endothelial activation.

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Haberzettl, Petra; Vladykovskaya, Elena; Srivastava, Sanjay; Bhatnagar, Aruni

2009-01-01

Acrolein is a ubiquitous environmental pollutant and an endogenous product of lipid peroxidation. It is also generated during the metabolism of several drugs and amino acids. In this study, we examined the effects of acrolein on endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with 2 to 10 microM acrolein led to an increase in the phosphorylation of eIF-2alpha within 10 to 30 min of exposure. This was followed by alternate splicing of XBP-1 mRNA and an increase in the expression of the endoplasmic reticulum (ER) chaperone genes Grp78 and Herp. Within 2-4 h of treatment, acrolein also increased the abundance and the nuclear transport of the transcription factors ATF3, AFT4, and CHOP. Acrolein-induced increase in ATF3 was prevented by treating the cells with the chemical chaperone - phenylbutyric acid (PBA). Treatment with acrolein increased phosphorylation of ERK1/2, p38, and JNK. The increase in JNK phosphorylation was prevented by PBA. Acrolein treatment led to activation and nuclear translocation of the transcription factor NF-kappaB and an increase in TNF-alpha, IL-6 and IL-8, but not MCP-1, mRNA. Increased expression of cytokine genes and NF-kappaB activation were not observed in cells treated with PBA. These findings suggest that exposure to acrolein induces ER stress and triggers the unfolded protein response and that NF-kappaB activation and stimulation of cytokine production by acrolein could be attributed, in part, to ER stress. Chemical chaperones of protein-folding may be useful in treating toxicological and pathological states associated with excessive acrolein exposure or production.

Role of endoplasmic reticulum stress in acrolein-induced endothelial activation

International Nuclear Information System (INIS)

Haberzettl, Petra; Vladykovskaya, Elena; Srivastava, Sanjay; Bhatnagar, Aruni

2009-01-01

Acrolein is a ubiquitous environmental pollutant and an endogenous product of lipid peroxidation. It is also generated during the metabolism of several drugs and amino acids. In this study, we examined the effects of acrolein on endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with 2 to 10 μM acrolein led to an increase in the phosphorylation of eIF-2α within 10 to 30 min of exposure. This was followed by alternate splicing of XBP-1 mRNA and an increase in the expression of the endoplasmic reticulum (ER) chaperone genes Grp78 and Herp. Within 2-4 h of treatment, acrolein also increased the abundance and the nuclear transport of the transcription factors ATF3, AFT4, and CHOP. Acrolein-induced increase in ATF3 was prevented by treating the cells with the chemical chaperone - phenylbutyric acid (PBA). Treatment with acrolein increased phosphorylation of ERK1/2, p38, and JNK. The increase in JNK phosphorylation was prevented by PBA. Acrolein treatment led to activation and nuclear translocation of the transcription factor NF-κB and an increase in TNF-α, IL-6 and IL-8, but not MCP-1, mRNA. Increased expression of cytokine genes and NF-κB activation were not observed in cells treated with PBA. These findings suggest that exposure to acrolein induces ER stress and triggers the unfolded protein response and that NF-κB activation and stimulation of cytokine production by acrolein could be attributed, in part, to ER stress. Chemical chaperones of protein-folding may be useful in treating toxicological and pathological states associated with excessive acrolein exposure or production

Mitochondrial enzymes and endoplasmic reticulum calcium stores as targets of oxidative stress in neurodegenerative diseases.

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Gibson, Gary E; Huang, Hsueh-Meei

2004-08-01

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation

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Shanks, Julia; Herring, Neil; Johnson, Errin; Liu, Kun; Li, Dan

2017-01-01

Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; Pneurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; Pneurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined. PMID:28223472

Endoplasmic reticulum remodeling tunes IP₃-dependent Ca²+ release sensitivity.

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Lu Sun

Full Text Available The activation of vertebrate development at fertilization relies on IP₃-dependent Ca²⁺ release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP₃ receptors within ER patches have a higher sensitivity to IP₃ than those in the neighboring reticular ER. The lateral diffusion rate of IP₃ receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP₃ receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP₃ receptors through ER remodeling is sufficient to sensitize IP₃-dependent Ca²⁺ release. Mathematical modeling supports this concept of 'geometric sensitization' of IP₃ receptors as a population, and argues that it depends on enhanced Ca²⁺-dependent cooperativity at sub-threshold IP₃ concentrations. This represents a novel mechanism of tuning the sensitivity of IP₃ receptors through ER remodeling during meiosis.

Endoplasmic Reticulum Stress-Associated Lipid Droplet Formation and Type II Diabetes

OpenAIRE

Zhang, Xuebao; Zhang, Kezhong

2012-01-01

Diabetes mellitus (DM), a metabolic disorder characterized by hyperglycemia, is caused by insufficient insulin production due to excessive loss of pancreatic β cells (type I diabetes) or impaired insulin signaling due to peripheral insulin resistance (type II diabetes). Pancreatic β cell is the only insulin-secreting cell type that has highly developed endoplasmic reticulum (ER) to cope with high demands of insulin synthesis and secretion. Therefore, ER homeostasis is crucial to the proper fu...

The Role of Endoplasmic Reticulum Stress in Diabetic Nephropathy.

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Fan, Ying; Lee, Kyung; Wang, Niansong; He, John Cijiang

2017-03-01

Diabetic nephropathy (DN) has become the leading cause of end-stage renal disease (ESRD) worldwide. Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development and progression of DN. Recent findings suggested that many attributes of DN, such as hyperglycemia, proteinuria, and increased advanced glycation end products and free fatty acids, can all trigger unfolded protein response (UPR) in kidney cells. Herein, we review the current knowledge on the role of ER stress in the setting of kidney injury with a specific emphasis on DN. As maladaptive ER stress response caused by excessively prolonged UPR will eventually cause cell death and increase kidney injury, several ER stress inhibitors have been shown to improve DN in animal models, albeit blocking both adaptive and maladaptive UPR. More recently, reticulon-1A (RTN1A), an ER-associated protein, was shown to be increased in both human and mouse diabetic kidneys. Its expression correlates with the progression of DN, and its polymorphisms are associated with kidney disease in people with diabetes. Increased RTN1A expression heightened the ER stress response and renal cell apoptosis, and conversely reduced RTN1A in renal cells decreased apoptosis and ameliorated kidney injury and DN progression, suggesting that RTN1A may be a novel target to specifically restrain the maladaptive UPR. These findings suggest that ER stress response in renal cells is a key driver of progression of DN and that the inhibition of the unchecked ER stress response in DN, such as by inhibition of RTN1A function, may be a promising therapeutic approach against DN.

circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

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Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

2017-12-13

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

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Stephanie Jamison

Full Text Available Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK in response to endoplasmic reticulum (ER stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A. Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2, to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting Endoplasmic Reticulum (ER) Stress Induced by Disturbed Flow.

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Chung, Jihwa; Kim, Kyoung Hwa; Lee, Seok Cheol; An, Shung Hyun; Kwon, Kihwan

2015-10-01

Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.

A postmortem study on indigestible foreign bodies in the rumen and reticulum of ruminants, eastern Ethiopia

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Seifu Negash

2015-05-01

Full Text Available A cross-sectional study was conducted on ruminants (cattle, sheep and goats slaughtered at Haramaya University and Haramaya municipal abattoirs from November 2013 to April 2014 in Haramaya, eastern Ethiopia. The objective of the study was to identify types and estimate the prevalence of foreign bodies in the rumen and reticulum of domestic ruminants in the area. From 810 randomly selected study animals, 422 (52.1% were found to have foreign bodies. Of the 332 cattle, 193 sheep and 285 goats examined, 144 (43.4%, 109 (56.5% and 169 (59.3% respectively were found with various types of foreign bodies. The prevalence of foreign bodies was significantly (χ2 = 17.53, p < 0.05 higher in sheep (59.3% and goats (56.7% than in cattle (43.4%. Overall the prevalence of foreign bodies in study animals with poor body condition was significantly higher (χ2 = 38.57, p < 0.05 than in those with medium and good body condition. A higher percentage of foreign bodies occurred in the rumen alone (87.9% than in the reticulum alone (5.0%, with the rest present in both. Significantly higher proportions of foreign bodies were observed in the rumen of cattle (χ2 = 332, p < 0.05, sheep (χ2 = 193, p < 0.05 and goats (χ2 = 285.0, p = 0.000 than in the reticulum. Plastic was the most commonly encountered (79.2% foreign body, followed by cloth (15.3% and rope (12.3%. In addition, metal (0.9% and calcified material and/or stone (1.0% were found in the reticulum of cattle. Lack of a plastic waste disposal system in the area as well as communal/free grazing of livestock in highly waste-polluted areas seemed to be major factors in the high occurrence of foreign bodies in ruminants. To change this, collaborative intervention schemes involving professionals, policy makers, livestock keepers and environmental activists are needed.

Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

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Tomasz J. Wodzicki

2015-01-01

Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

Met receptor inhibitor SU11274 localizes in the endoplasmic reticulum.

Science.gov (United States)

Wiest, Edwin J; Smith, Heather Jensen; Hollingsworth, Michael A

2018-07-02

We discovered that SU11274, a class I c-Met inhibitor, fluoresces when excited by 488 nm laser light and showed rapid specific accumulation in distinct subcellular compartments. Given that SU11274 reduces cancer cell viability, we exploited these newly identified spectral properties to determine SU11274 intracellular distribution and accumulation in human pancreatic cancer cells. The aim of the studies reported here was to identify organelle(s) to which SU11274 is trafficked. We conclude that SU11274 rapidly and predominantly accumulates in the endoplasmic reticulum. Copyright © 2018. Published by Elsevier Inc.

Endoplasmic reticulum involvement in yeast cell death

International Nuclear Information System (INIS)

Nicanor Austriaco, O.

2012-01-01

Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

Endoplasmic Reticulum (ER Stress and Endocrine Disorders

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Daisuke Ariyasu

2017-02-01

Full Text Available The endoplasmic reticulum (ER is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR, which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI, Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2 are discussed in this article.

Endoplasmic Reticulum (ER) Stress and Endocrine Disorders

Science.gov (United States)

Ariyasu, Daisuke; Yoshida, Hiderou; Hasegawa, Yukihiro

2017-01-01

The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the “unfolded protein response” (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article. PMID:28208663

Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane.

Science.gov (United States)

Savino, John A; Evans, Jodi F; Rabinowitz, Dorianne; Auborn, Karen J; Carter, Timothy H

2006-03-01

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.

Fish sarcoplasmic proteins as a high value marine material for wound dressing applications.

Science.gov (United States)

Vieira, Sara; Franco, Albina R; Fernandes, Emanuel M; Amorim, Sara; Ferreira, Helena; Pires, Ricardo A; Reis, Rui L; Martins, Albino; Neves, Nuno M

2018-07-01

Fish sarcoplasmic proteins (FSP) constitute around 25-30% of the total fish muscle protein. As the FSP are water soluble, FSP were isolated from fresh cod (Gadus morhua) by centrifugation. By SDS-PAGE, it was possible to determine the composition of FSP extracts (FSP-E). The FSP-E undergo denaturation at 44.12 ± 2.34° C, as characterized by differential scanning calorimetry thermograms (DSC). The secondary structure of FSP-E is mainly composed by α-helix structure, as determined by circular dichroism. The cytocompatibility of FSP-E, at concentrations ranging from 5 to 20 mg/mL, was investigated. Concentrations lower than 10 mg/mL have no cytotoxicity cultures of fibroblasts over 72 h. Further on, FSP membranes (FSP-M) were produced by spin coating to evaluate its properties. FSP-M shown having uniform surface as analyzed by Scanning Electron Microscopy (SEM). The relative amount of α-helix structures is higher when compared with the FSP-E. The FSP-M have higher temperature stability than the FSP-E, since they presented a denaturation temperature of 58.88 ± 3.36° C, according to the DSC analysis. FSP-M shown distinctive mechanical properties, with a stiffness of 16.57 ± 3.95 MPa and a yield strength of 23.85 ± 5.97 MPa. Human lung fibroblasts cell lines (MRC-5) were cultured in direct contact with FSP-M, demonstrating its cytocompatibility for 48 h. Based on these results, FSP can be considered a potential biomaterial recovered from nature, for wound dressing applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Endoplasmic Reticulum Stress and Autophagy in Homocystinuria Patients with Remethylation Defects.

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Ainhoa Martínez-Pizarro

Full Text Available Proper function of endoplasmic reticulum (ER and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP3R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2 were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca2+ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.

How are proteins reduced in the endoplasmic reticulum?

DEFF Research Database (Denmark)

Ellgaard, Lars; Sevier, Carolyn S.; Bulleid, Neil J.

2018-01-01

The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction...... of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here...... why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway....

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?

DEFF Research Database (Denmark)

Skagen, Peter Storgaard; Horn, T; Kruse, H A

2011-01-01

in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in "bundles" and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae...... close to mineralized "islands" in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co-localization within the islands of types I, II, X collagens...... and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non-conclusive, only single nucleotide...

Prospects of X-ray microanalysis in the study of pathophysiology of myocardial contraction

International Nuclear Information System (INIS)

Wendt-Gallitelli, M.F.; Schwegler, M.; Holubarsch, C.; Jacob, R.; Wolburg, H.; Schlote, W.

1980-01-01

X-ray microanalysis was used to compare chemically untreated cryosections of quick-frozen myocardial tissue in 'caffeine contracture' with cryosections of normal muscle. Our goal was to find out if it is possible by this method to detect changes in the calcium compartmentalization of the myocardial cell occurring by changes in its functional state. While it is possible to quantitate calcium in the cisternae of sarcoplasmic reticulum of the control muscle preparation, calcium could never be detected in these compartments of caffeine-contracted muscles. In active microsomal fraction of ventricular myocardium it is possible to quantitate calcium and also to distinguish two components on account of their different ability to accumulate this element. The calcium content is different in the two components of the fraction. (orig.) [de

Targeting Cardiomyocyte Ca2+ Homeostasis in Heart Failure

Science.gov (United States)

Røe, Åsmund T.; Frisk, Michael; Louch, William E.

2015-01-01

Improved treatments for heart failure patients will require the development of novel therapeutic strategies that target basal disease mechanisms. Disrupted cardiomyocyte Ca2+ homeostasis is recognized as a major contributor to the heart failure phenotype, as it plays a key role in systolic and diastolic dysfunction, arrhythmogenesis, and hypertrophy and apoptosis signaling. In this review, we outline existing knowledge of the involvement of Ca2+ homeostasis in these deficits, and identify four promising targets for therapeutic intervention: the sarcoplasmic reticulum Ca2+ ATPase, the Na+-Ca2+ exchanger, the ryanodine receptor, and t-tubule structure. We discuss experimental data indicating the applicability of these targets that has led to recent and ongoing clinical trials, and suggest future therapeutic approaches. PMID:25483944

Endoplasmic reticulum-dependent redox reactions control endoplasmic reticulum-associated degradation and pathogen entry.

Science.gov (United States)

Walczak, Christopher P; Bernardi, Kaleena M; Tsai, Billy

2012-04-15

Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host-pathogen interactions. Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions.

Transgenic plants with increased calcium stores

Science.gov (United States)

Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Robertson, Dominique (Inventor); Boss, Wendy (Inventor)

2004-01-01

The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

Endoplasmic reticulum transport of glutathione by Sec61 is regulated by Ero1 and Bip

DEFF Research Database (Denmark)

Ponsero, Alise J.; Igbaria, Aeid; Darch, Maxwell A.

2017-01-01

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative...... oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import...... by reduction, causing Bip oxidation and inhibition of glutathione transport. Coupling of glutathione ER import to Ero1 activation provides a basis for glutathione ER redox poise maintenance....

Dose-dependent effects of cisplatin on the severity of testicular injury in Sprague Dawley rats: reactive oxygen species and endoplasmic reticulum stress

Directory of Open Access Journals (Sweden)

Soni KK

2016-12-01

Full Text Available Kiran Kumar Soni,1 Hye Kyung Kim,2 Bo Ram Choi,1 Keshab Kumar Karna,1 Jae Hyung You,1 Jai Seong Cha,1 Yu Seob Shin,1 Sung Won Lee,3 Chul Young Kim,4 Jong Kwan Park1 1Department of Urology, Institute for Medical Sciences, Chonbuk National University Medical School – Biomedical Research and Institute and Clinical Trial Center for Medical Devices, Chonbuk National University Hospital, Jeonju, 2College of Pharmacy, Kyungsung University, Busan, 3Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University Medical School, Seoul, 4College of Pharmacy, Hanyang University, Ansan, Republic of Korea Abstract: Cisplatin (CIS is used in the treatment of cancer, but its nonspecific systemic actions lead to toxic effects on other parts of the body. This study investigated the severity of CIS toxicity by increasing its dose over a constant time period. Sprague Dawley rats were divided into five treatment groups and control group with CIS (2, 4, 6, 8, and 10 mg/kg administered intraperitoneally for 5 days. The body and organs were weighed, epididymal sperm was counted, and sperm motility and sperm apoptosis were evaluated. Blood samples were evaluated for complete blood count, reactive oxygen and nitrogen species, malondialdehyde levels, and total testosterone. The testicular tissue was examined for steroidogenic acute regulatory protein and endoplasmic reticulum stress protein. Epididymal sperm was collected for CatSper Western blot. The toxic effects of different doses of CIS on the testis and kidney were compared histologically. The weights of body, testis, epididymis, prostate, seminal vesicle, and kidney; sperm count; sperm motility; steroidogenic acute regulatory protein level; and epididymal sperm count were significantly lower in the CIS-treated groups than in the control group. In contrast, sperm apoptosis, plasma reactive oxygen and nitrogen species, and malondialdehyde, testosterone, red blood cell

Membrane junctions in Xenopus eggs: their distribution suggests a role in calcium regulation.

Science.gov (United States)

Gardiner, D M; Grey, R D

1983-04-01

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma membrane is associated with junctions) and increase dramatically during progesterone-induced maturation. Junctions in the mature, unactivated egg are two to three times more abundant in the animal hemisphere (25-30 percent of the plasma membrane associated with junction) as compared with the vegetal hemisphere (10-15 percent). Junction density decreases rapidly to values characteristic of immature oocytes in response to egg activation. The plasma membrane-ER junctions of xenopus eggs are strikingly similar in structure to membrane junctions in muscle cells thought to be essential in the triggering of intracellular calcium release from the sarcoplasmic reticulum. In addition, the junctions' distinctive, animal-vegetal polarity of distribution, their dramatic appearance during maturation, and their disapperance during activation are correlated with previously documented patterns of calcium-mediated events in anuran eggs. We discuss several lines of evidence supporting the hypothesis that these junctions in xenopus eggs are sites that transduce extracellular events into intracellular calcium release during fertilization and activation of development.

Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

OpenAIRE

Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A.; Salic, Adrian

2011-01-01

The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is s...

Erlotinib promotes endoplasmic reticulum stress-mediated injury in the intestinal epithelium

Energy Technology Data Exchange (ETDEWEB)

Fan, Lu; Hu, Lingna; Yang, Baofang; Fang, Xianying; Gao, Zhe; Li, Wanshuai; Sun, Yang; Shen, Yan; Wu, Xuefeng [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China); Shu, Yongqian [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029 (China); Gu, Yanhong, E-mail: guluer@163.com [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029 (China); Wu, Xudong, E-mail: xudongwu@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093 (China)

2014-07-01

Erlotinib, a popular drug for treating non-small cell lung cancer (NSCLC), causes diarrhea in approximately 55% of patients receiving this drug. In the present study, we found that erlotinib induced barrier dysfunction in rat small intestine epithelial cells (IEC-6) by increasing epithelial permeability and down-regulating E-cadherin. The mRNA levels of various pro-inflammatory cytokines (Il-6, Il-25 and Il-17f) were increased after erlotinib treatment in IEC-6 cells. Erlotinib concentration- and time-dependently induced apoptosis and endoplasmic reticulum (ER) stress in both IEC-6 and human colon epithelial cells (CCD 841 CoN). Intestinal epithelial injury was also observed in male C57BL/6J mice administrated with erlotinib. Knockdown of C/EBP homologous protein (CHOP) with small interference RNA partially reversed erlotinib-induced apoptosis, production of IL-6 and down-regulation of E-cadherin in cultured intestinal epithelial cells. In conclusion, erlotinib caused ER stress-mediated injury in the intestinal epithelium, contributing to its side effects of diarrhea in patients. - Highlights: • Erlotinib destroyed barrier integrity both in vitro and in vivo. • Erlotinib induced inflammation both in vitro and in vivo. • Erlotinib induced apoptosis both in vitro and in vivo. • ER stress contributed to erlotinib-induced barrier dysfunction.

Erlotinib promotes endoplasmic reticulum stress-mediated injury in the intestinal epithelium

International Nuclear Information System (INIS)

Fan, Lu; Hu, Lingna; Yang, Baofang; Fang, Xianying; Gao, Zhe; Li, Wanshuai; Sun, Yang; Shen, Yan; Wu, Xuefeng; Shu, Yongqian; Gu, Yanhong; Wu, Xudong; Xu, Qiang

2014-01-01

Erlotinib, a popular drug for treating non-small cell lung cancer (NSCLC), causes diarrhea in approximately 55% of patients receiving this drug. In the present study, we found that erlotinib induced barrier dysfunction in rat small intestine epithelial cells (IEC-6) by increasing epithelial permeability and down-regulating E-cadherin. The mRNA levels of various pro-inflammatory cytokines (Il-6, Il-25 and Il-17f) were increased after erlotinib treatment in IEC-6 cells. Erlotinib concentration- and time-dependently induced apoptosis and endoplasmic reticulum (ER) stress in both IEC-6 and human colon epithelial cells (CCD 841 CoN). Intestinal epithelial injury was also observed in male C57BL/6J mice administrated with erlotinib. Knockdown of C/EBP homologous protein (CHOP) with small interference RNA partially reversed erlotinib-induced apoptosis, production of IL-6 and down-regulation of E-cadherin in cultured intestinal epithelial cells. In conclusion, erlotinib caused ER stress-mediated injury in the intestinal epithelium, contributing to its side effects of diarrhea in patients. - Highlights: • Erlotinib destroyed barrier integrity both in vitro and in vivo. • Erlotinib induced inflammation both in vitro and in vivo. • Erlotinib induced apoptosis both in vitro and in vivo. • ER stress contributed to erlotinib-induced barrier dysfunction

Model of Ca(2+) Concentration Controlled by Sarcoplasmic Reticulum of Skeletal Muscle, Using the State Transition

National Research Council Canada - National Science Library

Yokota, M

2001-01-01

...). This report proposed a model that represents Ca(2+) in a muscle cell controlled by the SR using a state transition probability model in which one state means that protein in the SR is binding ligands, and the other...

Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

Science.gov (United States)

Smith, Jennifer L; Reloj, Allison R; Nataraj, Parvathi S; Bartos, Daniel C; Schroder, Elizabeth A; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Anderson, Corey L; January, Craig T; Delisle, Brian P

2013-11-01

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

Cucurbita ficifolia Bouché increases insulin secretion in RINm5F cells through an influx of Ca(2+) from the endoplasmic reticulum.

Science.gov (United States)

Miranda-Perez, Maria Elizabeth; Ortega-Camarillo, Clara; Del Carmen Escobar-Villanueva, Maria; Blancas-Flores, Gerardo; Alarcon-Aguilar, Francisco Javier

2016-07-21

Cucurbita ficifolia Bouché(C. ficifolia) is a plant used in Mexican traditional medicine to control type 2 diabetes (T2D). The hypoglycemic effect of the fruit of C. ficifolia has been demonstrated in different experimental models and in T2D patients. It has been proposed that D-chiro-inositol (DCI) is the active compound of the fruit. Additionally, it has been reported that C. ficifolia increases the mRNA expression of insulin and Kir 6.2 (a component of the ATP-sensitive potassium (K(+)ATP) channel, which is activated by sulphonylurea) in RINm5F cells. However, it remains unclear whether C. ficifolia and DCI causes the secretion of insulin by increasing the concentration of intracellular calcium ([Ca(2+)]i) through K(+)ATP channel blockage or from the reservoir in the endoplasmic reticulum (ER). The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic β-cells were incubated with different concentrations (50, 100, 200 and 400μM) of DCI alone or C. ficifolia (9, 18, 36 and 72µg of extract/mL), and the [Ca(2+)]i of the cells was quantified. The cells were preloaded with the Ca(2+) fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K(+)ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia. It was found that DCI alone did not increase [Ca(2+)]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca(2+)]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca(2+)]i by 50%, while insulin secretion

Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness

DEFF Research Database (Denmark)

Gill, David J; Tham, Keit Min; Chia, Joanne

2013-01-01

Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-a...

Impact of high cholesterol and endoplasmic reticulum stress on metabolic diseases: An updated mini-review

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Erdi Sozen

2017-08-01

Full Text Available Endoplasmic reticulum (ER is the major site of protein folding and calcium storage. Beside the role of ER in protein homeostasis, it controls the cholesterol production and lipid-membrane biosynthesis as well as surviving and cell death signaling mechanisms in the cell. It is well-documented that elevated plasma cholesterol induces adverse effects in cardiovascular diseases (CVDs, liver disorders, such as non-alcoholic fatty liver disease (NAFLD, non-alcoholic steatosis hepatitis (NASH, and metabolic diseases which are associated with oxidative and ER stress. Recent animal model and human studies have showed high cholesterol and ER stress as an emerging factors involved in the development of many metabolic diseases. In this review, we will summarize the crucial effects of hypercholesterolemia and ER stress response in the pathogenesis of CVDs, NAFLD/NASH, diabetes and obesity which are major health problems in western countries. Keywords: Endoplasmic reticulum stress, High cholesterol, Cardiovascular diseases, Non-alcoholic fatty liver disease, Non-alcoholic steatosis hepatitis

Acrolein Disrupts Tight Junction Proteins and Causes Endoplasmic Reticulum Stress-Mediated Epithelial Cell Death Leading to Intestinal Barrier Dysfunction and Permeability.

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Chen, Wei-Yang; Wang, Min; Zhang, Jingwen; Barve, Shirish S; McClain, Craig J; Joshi-Barve, Swati

2017-12-01

Increasing evidence suggests that environmental and dietary factors can affect intestinal epithelial integrity leading to gut permeability and bacterial translocation. Intestinal barrier dysfunction is a pathogenic process associated with many chronic disorders. Acrolein is an environmental and dietary pollutant and a lipid-derived endogenous metabolite. The impact of acrolein on the intestine has not been investigated before and is evaluated in this study, both in vitro and in vivo. Our data demonstrate that oral acrolein exposure in mice caused damage to the intestinal epithelial barrier, resulting in increased permeability and subsequently translocation of bacterial endotoxin-lipopolysaccharide into the blood. Similar results were seen in vitro using established Caco-2 cell monolayers wherein acrolein decreased barrier function and increased permeability. Acrolein also caused the down-regulation and/or redistribution of three representative tight junction proteins (ie, zonula occludens-1, Occludin, Claudin-1) that critically regulate epithelial paracellular permeability. In addition, acrolein induced endoplasmic reticulum stress-mediated death of epithelial cells, which is an important mechanism contributing to intestinal barrier damage/dysfunction, and gut permeability. Overall, we demonstrate that exposure to acrolein affects the intestinal epithelium by decrease/redistribution of tight junction proteins and endoplasmic reticulum stress-mediated epithelial cell death, thereby resulting in loss of barrier integrity and function. Our findings highlight the adverse consequences of environmental and dietary pollutants on intestinal barrier integrity/function with relevance to gut permeability and the development of disease. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Endoplasmic Reticulum Stress in Reproductive Function

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Kang-sheng LIU

2016-09-01

Full Text Available Normal folding requires that unique conditions should be maintained within the endoplasmic reticulum (ER lumen, and nascent proteins are initially bound to Ca2+dependent chaperone proteins. Proteins synthesized in the ER are properly folded with the assistance of ER chaperones. misfolded proteins are disposed by ER-associated protein degradation. Accumulation of misfolded proteins in the ER triggers an adaptive ER stress response, which leads to activation of the unfolded protein response (UPR, a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cells. It has been shown that ER stress involves in pathophysiological development of many diseases, including neurological diseases. However, nowadays, a few studies have begun to focus on the possibility that the accumulation of misfolded proteins can also contribute to reproductive diseases. In this article, we mainly introduced the involvement of ER stress response in preimplantation embryos, placental development, intrauterine growth restriction (IUGR and testicular germ cells so as to provide important insights for the molecular mechanisms of ER stress-induced apoptosis in reproductive diseases.

T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

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Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

2015-02-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long-chain bases from sea cucumber mitigate endoplasmic reticulum stress and inflammation in obesity mice

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Shiwei Hu

2017-07-01

Full Text Available Endoplasmic reticulum (ER stress and inflammation can induce hyperglycemia. Long-chain bases (LCBs from sea cucumber exhibit antihyperglycemic activities. However, their effects on ER stress and inflammation are unknown. We investigated the effects of LCBs on ER stress and inflammatory response in high-fat, fructose diet-induced obesity mice. Reactive oxygen species and free fatty acids were measured. Inflammatory cytokines in serum and their mRNA expressions in epididymal adipose tissues were investigated. Hepatic ER stress-related key genes were detected. c-Jun NH2-terminal kinase and nuclear factor κB inflammatory pathways were also evaluated in the liver. Results showed that LCBs reduced serum and hepatic reactive oxygen species and free fatty acids concentrations. LCBs decreased serum proinflammatory cytokines levels, namely interleukin (IL-1β, tumor necrosis factor-α, IL-6, macrophage inflammatory protein 1, and c-reactive protein, and increased anti-inflammatory cytokine IL-10 concentration. The mRNA and protein expressions of these cytokines in epididymal adipose tissues were regulated by LCBs as similar to their circulatory contents. LCBs inhibited phosphorylated c-Jun NH2-terminal kinase and inhibitor κ kinase β, and nuclear factor κB nuclear translocation. LCBs also inhibited mRNA expression of ER stress markers glucose regulated protein, activating transcription factor 6, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase, and X-box binding protein 1, and phosphorylation of eukaryotic initiation factor-α and inositol requiring enzyme 1α. These results indicate that LCBs can alleviate ER stress and inflammatory response. Nutritional supplementation with LCBs may offer an adjunctive therapy for RE stress-associated inflammation.

CDIP1-BAP31 Complex Transduces Apoptotic Signals from Endoplasmic Reticulum to Mitochondria under Endoplasmic Reticulum Stress

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Takushi Namba

2013-10-01

Full Text Available Resolved endoplasmic reticulum (ER stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a proapoptotic p53 target, CDIP1, acts as a key signal transducer of ER-stress-mediated apoptosis. We identify B-cell-receptor-associated protein 31 (BAP31 as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent truncated Bid (tBid and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER-stress-mediated apoptosis. Altogether, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a mechanism for establishing an ER-mitochondrial crosstalk for ER-stress-mediated apoptosis signaling.

Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread.

Science.gov (United States)

Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Wang, JiaJia; Li, Cunzhi; Guo, ShiJun; Li, Lin; Hu, Song-Qing

2018-08-01

This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

Science.gov (United States)

Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

2018-01-01

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

Quantification of plasmodesmatal endoplasmic reticulum coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching

DEFF Research Database (Denmark)

Martens, Helle; Roberts, Alison G.; Oparka, Karl J.

2006-01-01

retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome......Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein...... and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues...

SH Oxidation Stimulates Calcium Release Channels (Ryanodine Receptors From Excitable Cells

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CECILIA HIDALGO

2000-01-01

Full Text Available The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub µM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed

Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection [version 2; referees: 1 approved, 3 approved with reservations

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Peter Wild

2018-02-01

Full Text Available Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1 or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1 by transmission and scanning electron microscopy, employing freezing technique protocols. Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the cis face. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii the process taking place at the outer nuclear

A lentivirally delivered photoactivatable GFP to assess continuity in the endoplasmic reticulum of neurones and glia

Czech Academy of Sciences Publication Activity Database

Jones, V. C.; Rodríguez Arellano, Jose Julio; Verkhratsky, Alexei; Jones, O. T.

2009-01-01

Roč. 458, č. 4 (2009), s. 809-818 ISSN 0031-6768 R&D Projects: GA ČR GA305/08/1384 Institutional research plan: CEZ:AV0Z50390512 Keywords : endoplasmic reticulum * calcium store * neurone Subject RIV: FH - Neurology Impact factor: 3.695, year: 2009

Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes

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Devasena Ponnalagu

2016-06-01

Full Text Available Chloride intracellular channel (CLICs proteins show 60–70% sequence identity to each other, and exclusively localize to the intracellular organelle membranes and cytosol. In support of our recent publication, “Molecular identity of cardiac mitochondrial chloride intracellular channel proteins” (Ponnalagu et al., 2016 [1], it was important to characterize the specificity of different CLIC paralogs/ortholog (CLIC1, CLIC4, CLIC5 and DmCLIC antibodies used to decipher their localization in cardiac cells. In addition, localization of CLICs in the other organelles such as endoplasmic reticulum (ER of cardiomyocytes was established. This article also provides data on the different primers used to show the relative abundance of CLIC paralogs in cardiac tissue and the specificity of the various CLIC antibodies used. We demonstrate that the predominant CLICs in the heart, namely CLIC1, CLIC4 and CLIC5, show differential distribution in endoplasmic reticulum. CLIC1 and CLIC4 both show co-localization to the endoplasmic reticulum whereas CLIC5 does not.

AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.

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Preissler, Steffen; Rato, Cláudia; Chen, Ruming; Antrobus, Robin; Ding, Shujing; Fearnley, Ian M; Ron, David

2015-12-17

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

Tc-99m pyrophosphate scanning after ischemic exercise in McArdle's disease

International Nuclear Information System (INIS)

Uno, Hideaki; Kawano, Keizo; Yukawa, Susumu; Nomoto, Hiroshi

1982-01-01

In order to clarify the mechanism of muscle contracture induced by ischemic exercise in a patient with McArdle's disease, bone scanning with Tc-99m pyrophosphate was performed. The clinical diagnosis was established in the patient based on the biochemical examinations of skeletal muscle biopsy. Ischemic exercise was done initially on the left forearm and then 20 hours later on the right forearm. Two hours later, 15 mCi of Tc-99m pyrophosphate was infused through the left antecubital vein. Exactly 4 hours later, a conventional bone scanning was carried out. In the patient with McArdle's disease, muscle contracture developed in both forearms during the ischemic exercise. Conventional bone scanning showed increased Tc-99m pyrophosphate labeling of the right forearm muscles at 2 hours after ischemic exercise. However, increased labeling of the left forearm muscles was not found at 22 hours after ischemic exercise. In the control, no muscle contracture developed during ischemic exercise and bone scan showed no increase in Tc-99m pyrophosphate labeling in the antebrachial region. These findings suggest that the basis of muscle contracture appears to be an increased concentration of Ca in muscle cells due to a failure of sarcoplasmic reticulum to reaccumulate Ca at ischemic exercise. (author)

The integrated endoplasmic reticulum stress response in Leishmania amazonensis macrophage infection: the role of X-box binding protein 1 transcription factor.

Science.gov (United States)

Dias-Teixeira, Karina Luiza; Calegari-Silva, Teresa Cristina; dos Santos, Guilherme R R M; Vitorino Dos Santos, José; Lima, Carolina; Medina, Jorge Mansur; Aktas, Bertal Huseyin; Lopes, Ulisses G

2016-04-01

Endoplasmic reticulum (ER) stress triggers the integrated ER-stress response (IERSR) that ensures cellular survival of ER stress and represents a primordial form of innate immunity. We investigated the role of IERSR duringLeishmania amazonensisinfection. Treatment of RAW 264.7 infected macrophages with the ER stress-inducing agent thapsigargin (TG; 1 μM) increasedL. amazonensisinfectivity in an IFN1-α receptor (IFNAR)-dependent manner. In Western blot assays, we showed thatL. amazonensisactivates the inositol-requiring enzyme (IRE1)/ X-box binding protein (XBP)-1-splicing arms of the IERSR in host cells. In chromatin immunoprecipitation (ChIP) assays, we showed an increased occupancy of enhancer and promoter sequences for theIfnbgene by XBP1 in infected RAW 264.7 cells. Knocking down XBP1 expression by transducing RAW 264.7 cells with the short hairpin XBP1 lentiviral vector significantly reduced the parasite proliferation associated with impaired translocation of phosphorylated IFN regulatory transcription factor (IRF)-3 to the nucleus and a decrease in IFN1-β expression. Knocking down XBP1 expression also increased NO concentration, as determined by Griess reaction and reduced the expression of antioxidant genes, such as heme oxygenase (HO)-1, that protect parasites from oxidative stress. We conclude thatL. amazonensisactivation of XBP1 plays a critical role in infection by protecting the parasites from oxidative stress and increasing IFN1-β expression.-Dias-Teixeira, K. L., Calegari-Silva, T. C., Dos Santos, G. R. R. M., Vitorino dos Santos, J., Lima, C., Medina, J. M., Aktas, B. H., Lopes, U. G. The integrated endoplasmic reticulum stress response inLeishmania amazonensismacrophage infection: the role of X-box binding protein 1 transcription factor. © FASEB.

Effect of Entodinium caudatum on starch intake and glycogen formation by Eudiplodinium maggii in the rumen and reticulum.

Science.gov (United States)

Bełżecki, Grzegorz; McEwan, Neil R; Kowalik, Barbara; Michałowski, Tadeusz; Miltko, Renata

2017-02-01

This study aimed to quantify the engulfed starch and reserve α-glucans (glycogen) in the cells of the ciliates Eudiplodinium maggii, as well the α-glucans in defaunated and selectively faunated sheep. The content of starch inside the cell of ciliates varied from 21 to 183mg/g protozoal DM relative to the rumen fauna composition whereas, the glycogen fluctuated between 17 and 126mg/g dry matter (DM) of this ciliate species. Establishment of the population Entodinium caudatum in the rumen of sheep already faunated with E. maggii caused a drop in both types of quantified carbohydrates. The content of α-glucans in the rumen of defaunated sheep varied from 4.4 to 19.9mg/g DM and increased to 7.4-29.9 or 11.8-33.9mg/g DM of rumen contents in the presence of only E. maggii or E. maggii and E. caudatum, respectively. The lowest content of the carbohydrates was always found just before feeding and the highest at 4h thereafter. The α-glucans in the reticulum varied 7.5-40.1, 14.3-76.8 or 21.9-106.1mg/g DM of reticulum content for defaunated, monofaunated or bifaunated sheep, respectively. The results indicated that both ciliate species engulf starch granules and convert the digestion products to the glycogen, diminishing the pool of starch available for amylolytic bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

Endoplasmic reticulum calcium transport ATPase expression during differentiation of colon cancer and leukaemia cells

International Nuclear Information System (INIS)

Papp, Bela; Brouland, Jean-Philippe; Gelebart, Pascal; Kovacs, Tuende; Chomienne, Christine

2004-01-01

The calcium homeostasis of the endoplasmic reticulum (ER) is connected to a multitude of cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Calcium is accumulated in the ER by various biochemically distinct sarco/endoplasmic reticulum calcium transport ATPase isoenzymes (SERCA isoforms). Experimental data indicate that the SERCA composition of some carcinoma and leukaemia cell types undergoes significant changes during differentiation, and that this is accompanied by modifications of SERCA-dependent calcium accumulation in the ER. Because ER calcium homeostasis can also influence cell differentiation, we propose that the modulation of the expression of various SERCA isoforms, and in particular, the induction of the expression of SERCA3-type proteins, is an integral part of the differentiation program of some cancer and leukaemia cell types. The SERCA content of the ER may constitute a new parameter by which the calcium homeostatic characteristics of the organelle are adjusted. The cross-talk between ER calcium homeostasis and cell differentiation may have some implications for the better understanding of the signalling defects involved in the acquisition and maintenance of the malignant phenotype

Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

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Denamur, Sophie; Boland, Lidvine [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Beyaert, Maxime [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Verstraeten, Sandrine L. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Fillet, Marianne [University of Liege, CIRM, Department of Pharmacy, Laboratory for the Analysis of Medicines, Quartier Hopital, Avenue Hippocrate, 15, B36, Tower 4, 4000 Liège 1 (Belgium); Tulkens, Paul M. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Bontemps, Françoise [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Mingeot-Leclercq, Marie-Paule [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium)

2016-10-15

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

International Nuclear Information System (INIS)

Denamur, Sophie; Boland, Lidvine; Beyaert, Maxime; Verstraeten, Sandrine L.; Fillet, Marianne; Tulkens, Paul M.; Bontemps, Françoise; Mingeot-Leclercq, Marie-Paule

2016-01-01

Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

Bridging the myoplasmic gap II: more recent advances in skeletal muscle excitation-contraction coupling.

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Bannister, Roger A

2016-01-01

In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca(2+) channel (Ca(V)1.1) in the plasma membrane to the channel pore of the type 1 ryanodine receptor (RyR1) nearly 10 nm away in the membrane of the sarcoplasmic reticulum (SR). Even though the roles of Ca(V)1.1 and RyR1 as voltage sensor and SR Ca(2+) release channel, respectively, have been established for nearly 25 years, the mechanism underlying communication between these two channels remains undefined. In the course of this article, I will review current viewpoints on this topic with particular emphasis on recent studies. © 2016. Published by The Company of Biologists Ltd.

Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration.

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Bernard-Marissal, Nathalie; Médard, Jean-Jacques; Azzedine, Hamid; Chrast, Roman

2015-04-01

Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1(-/-) mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum-mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Vildagliptin preserves the mass and function of pancreatic β cells via the developmental regulation and suppression of oxidative and endoplasmic reticulum stress in a mouse model of diabetes

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Hamamoto, S; Kanda, Y; Shimoda, M; Tatsumi, F; Kohara, K; Tawaramoto, K; Hashiramoto, M; Kaku, K

2013-01-01

Aim We investigated the molecular mechanisms by which vildagliptin preserved pancreatic β cell mass and function. Methods Morphological, biochemical and gene expression profiles of the pancreatic islets were investigated in male KK-Ay-TaJcl(KK-Ay) and C57BL/6JJcl (B6) mice aged 8 weeks which received either vildagliptin or a vehicle for 4 weeks. Results Body weight, food intake, fasting blood glucose, plasma insulin and active glucagon-like peptide-1 were unchanged with vildagliptin treatment in both mice. In KK-Ay mice treated with vildagliptin, increased plasma triglyceride (TG) level and islet TG content were decreased, insulin sensitivity significantly improved, and the glucose tolerance ameliorated with increases in plasma insulin levels. Furthermore, vildagliptin increased glucose-stimulated insulin secretion, islet insulin content and pancreatic β cell mass in both strains. By vildagliptin, the expression of genes involved in cell differentiation/proliferation was upregulated in both strains, those related to apoptosis, endoplasmic reticulum stress and lipid synthesis was decreased and those related to anti-apoptosis and anti-oxidative stress was upregulated, in KK-Ay mice. The morphological results were consistent with the gene expression profiles. Conclusion Vildagliptin increases β cell mass by not only directly affecting cell kinetics but also by indirectly reducing cell apoptosis, oxidative stress and endoplasmic reticulum stress in diabetic mice. PMID:22950702

Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

International Nuclear Information System (INIS)

Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

2015-01-01

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl 4 )-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl 4 -treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl 4 -treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl 4 -treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl 4 , presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity

Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

Energy Technology Data Exchange (ETDEWEB)

Harris, Todd R. [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Bettaieb, Ahmed [Department of Nutrition, University of California, Davis, CA 95616 (United States); Kodani, Sean; Dong, Hua [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Myers, Richard; Chiamvimonvat, Nipavan [Department of Internal Medicine: Cardiovascular, University of California, Davis, CA 95616 (United States); Haj, Fawaz G. [Department of Nutrition, University of California, Davis, CA 95616 (United States); Department of Internal Medicine: Endocrinology, Diabetes and Metabolism, University of California, Davis, CA 95616 (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)

2015-07-15

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

Renal endoplasmic reticulum stress is coupled to impaired autophagy in a mouse model of GSD Ia.

Science.gov (United States)

Farah, Benjamin L; Landau, Dustin J; Wu, Yajun; Sinha, Rohit A; Loh, Alwin; Bay, Boon-Huat; Koeberl, Dwight D; Yen, Paul M

2017-11-01

GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia. Copyright © 2017 Elsevier Inc. All rights reserved.

Tributyltin-induced endoplasmic reticulum stress and its Ca(2+)-mediated mechanism.

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Isomura, Midori; Kotake, Yaichiro; Masuda, Kyoichi; Miyara, Masatsugu; Okuda, Katsuhiro; Samizo, Shigeyoshi; Sanoh, Seigo; Hosoi, Toru; Ozawa, Koichiro; Ohta, Shigeru

2013-10-01

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Roles of endoplasmic reticulum stress and unfolded protein response associated genes in seed stratification and bud endodormancy during chilling accumulation in Prunus persica.

Directory of Open Access Journals (Sweden)

Xi Ling Fu

Full Text Available Dormancy mechanisms in seeds and buds arrest growth until environmental conditions are optimal for development. A genotype-specific period of chilling is usually required to release dormancy, but the underlying molecular mechanisms are still not fully understood. To discover transcriptional pathways associated with dormancy release common to seed stratification and bud endodormancy, we explored the chilling-dependent expression of 11 genes involved in endoplasmic reticulum stress and the unfolded protein response signal pathways. We propose that endoplasmic reticulum stress and the unfolded protein response impact on seed as well as bud germination and development by chilling-dependent mechanisms. The emerging discovery of similarities between seed stratification and bud endodormancy status indicate that these two processes are probably regulated by common endoplasmic reticulum stress and unfolded protein response signalling pathways. Clarification of regulatory pathways common to both seed and bud dormancy may enhance understanding of the mechanisms underlying dormancy and breeding programs may benefit from earlier prediction of chilling requirements for uniform blooming of novel genotypes of deciduous fruit tree species.

Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms.

Science.gov (United States)

Yu, Zhi-Bin

2013-11-01

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high-frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus (EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase (nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated

Endoplasmic reticulum stress causes EBV lytic replication.

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Taylor, Gwen Marie; Raghuwanshi, Sandeep K; Rowe, David T; Wadowsky, Robert M; Rosendorff, Adam

2011-11-17

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2

International Nuclear Information System (INIS)

Ascher, David B.; Polekhina, Galina; Parker, Michael W.

2012-01-01

The luminal domain of human endoplasmic reticulum aminopeptidase 2 has been expressed, purified and crystallized. The crystals belonged to the orthorhombic space group P2 1 2 1 2 and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house source. Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures

Computed tomography of the abdomen in Saanen goats: I.Reticulum, rumen and omasum

International Nuclear Information System (INIS)

Braun, U.; Irmer, M.; Augsburger, H.; Jud, R.; Ohlert, S.

2011-01-01

Computed tomography (CT) of the reticulum, rumen and omasum was carried out in 30 healthy goats and the images were compared to corresponding body sections obtained at postmortem. A multidetector CT was used to examine goats in sternal recumbency. A setting of 120 KV and 270 mA was used to produce 1.5-mm transverse slices from the fifth thoracic vertebra to the sacrum. Soft tissue structures were assessed in a soft tissue with a window width (W) of 400 Hounsfield Units (HU), and a window level (L) of 40 HU. The layering of the ruminal contents was assessed in an ingesta window with a W of 1500 HU and an L of 30 HU. After subjective evaluation, the size of the rumen and omasum, the thickness of the walls of the reticulum, rumen and omasum and the height of the gas cap and fibre and liquid phases of the rumen were measured. Fifteen goats were euthanised after CT examination, placed in sternal recumbency and frozen at -18 єC for three to 10 days. Thirteen goats were then cut into 1.0- to 1.5-cm-thick transverse slices. One goat was cut in dorsal-plane slices and another in sagittal slices. The structures in the CT images were identified by using the corresponding anatomical slices

VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

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Shih, Yu-Tzu; Hsueh, Yi-Ping

2016-03-17

Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

Peptide YY (3-36) modulates intracellular calcium through activation of the phosphatidylinositol pathway in hippocampal neurons.

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Domingues, Michelle Flores; de Assis, Dênis Reis; Piovesan, Angela Regina; Belo, Cháriston André Dal; da Costa, Jaderson Costa

2018-02-01

Peptide YY (PYY) belongs to the neuropeptide Y (NPY) family, which also includes the pancreatic polypeptide (PP) and NPY. PYY is secreted by the intestinal L cells, being present in the blood stream in two active forms capable of crossing the blood brain barrier, PYY (1-36) and its cleavage product, PYY (3-36). PYY is a selective agonist for the Y2 receptor (Y2R) and these receptors are abundant in the hippocampus. Here we investigated the mechanisms by which PYY (3-36) regulates intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in hippocampal neurons by employing a calcium imaging technique in hippocampal cultures. Alterations in [Ca 2+ ] i were detected by changes in the Fluo-4 AM reagent emission. PYY (3-36) significantly increased [Ca 2+ ] from the concentration of 10 -11 M as compared to the controls (infusion of HEPES-buffered solution (HBS) solution alone). The PYY (3-36)-increase in [Ca 2+ ] i remained unchanged even in Ca 2+ -free extracellular solutions. Sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase pump (SERCA pump) inhibition partially prevent the PYY (3-36)-increase of [Ca 2+ ] i and inositol 1,4,5-triphosphate receptor (IP3R) inhibition also decreased the PYY (3-36)-increase of [Ca 2+ ] i . Taken together, our data strongly suggest that PYY (3-36) mobilizes calcium from the neuronal endoplasmic reticulum (ER) stores towards the cytoplasm. Next, we showed that PYY (3-36) inhibited high K + -induced increases of [Ca 2+ ] i , suggesting that PYY (3-36) could also act by activating G-protein coupled inwardly rectifying potassium K + channels. Finally, the co-infusion of the Y2 receptor (Y2R) antagonist BIIE0246 with PYY (3-36) abolished the [Ca 2+ ] i increase induced by the peptide, suggesting that PYY (3-36)-induced [Ca 2+ ] i increase in hippocampal neurons occurs via Y2Rs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in amino transferases and muscle proteins when treating pigmeat with ionising rays

Energy Technology Data Exchange (ETDEWEB)

Hamm, R; Hofmann, K; Gruenewald, T; Partmann, W [Bundesanstalt fuer Fleischforschung, Kulmbach (F.R. Germany). Inst. fuer Chemie und Physik; Bundesforschungsanstalt fuer Ernaehrung, Karlsruhe [F.R. Germany

1975-01-01

Slices of lean pigmeat were treated with electron beams doses of 0.2, 1.0 and 5.0 Mrad. Low irradiation doses led to an increase in the activity of aspartate amino transferase (GOT) and alanin amino transferase (GPT) in the tissue generally and in the sarcoplasm (juice expressed from the muscle). 5 Mrad caused a great reduction in the activity of GOT and GPT in the tissue and the sarcoplasm. It seems doubtful whether this inactivation is due to a destruction of enzyme sulphhydryl groups. Irradiating with 5 Mrad resulted in partial release of the mitochondrial GOT isozyme (GOTsub(M)) into the sarcoplasm. This indicates damage to the mitochondrial membranes by ionising radiation. Irradiating the pigmeat increased the pH of the tissue and lowered its water binding ability (increase in drip). Up to a dose of 1 Mrad the solubility of the sarcoplasmic proteins was not definitely affected, but 5 Mrad caused a considerable drop in protein solubility. Surprisingly a dose of even 5 Mrad did not change the total number of sulphhydryl groups present in the tissue. Sephadex thin layer electrophoresis showed that at 0.2 Mrad there was a drastic decrease in the myosin band and an increase in peptide fragments of low molecular weight, whilst actin was little changed.

Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast

DEFF Research Database (Denmark)

Hou, Jin; Tang, Hongting; Liu, Zihe

2014-01-01

In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects...... the cell from stresses. Here, we find that a constitutive activation of the HSR could increase ER stress resistance in both wild-type and UPR-deficient cells. Activation of HSR decreased UPR activation in the WT (as shown by the decreased HAC1 mRNA splicing). We analyzed the genome-wide transcriptional...... response in order to propose regulatory mechanisms that govern the interplay between UPR and HSR and followed up for the hypotheses by experiments in vivo and in vitro. Interestingly, we found that the regulation of ER stress response via HSR is (1) only partially dependent on over-expression of Kar2p (ER...

The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

Science.gov (United States)

Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica

2014-03-01

Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

Heme oxygenase-1 comes back to endoplasmic reticulum

Energy Technology Data Exchange (ETDEWEB)

Kim, Hong Pyo [School of Biological Sciences, Ulsan University (Korea, Republic of); Pae, Hyun-Ock [Department of Immunology, Wonkwang University School of Medicine (Korea, Republic of); Back, Sung Hun; Chung, Su Wol [School of Biological Sciences, Ulsan University (Korea, Republic of); Woo, Je Moon [Department of Opthalmology, Ulasn University Hospital (Korea, Republic of); Son, Yong [Department of Anesthesiology and Pain Medicine, Wonkwang University School of Medicine (Korea, Republic of); Chung, Hun-Taeg, E-mail: chung@ulsan.ac.kr [School of Biological Sciences, Ulsan University (Korea, Republic of)

2011-01-07

Research highlights: {yields} Although multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. {yields} HO-1 expression at ER is induced by a diverse set of conditions that cause ER stressors. {yields} CO may induce HO-1 expression in human ECs by activating Nrf2 through PERK phosphorylation in a positive-feedback manner. {yields} ER-residing HO-1 and its cytoprotective activity against ER stress is discussed. -- Abstract: Originally identified as a rate-limiting enzyme for heme catabolism, heme oxygenase-1 (HO-1) has expanded its roles in anti-inflammation, anti-apoptosis and anti-proliferation for the last decade. Regulation of protein activity by location is well appreciated. Even though multiple compartmentalization of HO-1 has been documented, the functional implication of this enzyme at these subcellular organelles is only partially elucidated. In this review we discuss the endoplasmic reticulum (ER)-residing HO-1 and its cytoprotective activity against ER stress.

Inhibitory Effects of Simvastatin on Oxidized Low-Density Lipoprotein-Induced Endoplasmic Reticulum Stress and Apoptosis in Vascular Endothelial Cells.

Science.gov (United States)

Zhang, Guo-Qiang; Tao, Yong-Kang; Bai, Yong-Ping; Yan, Sheng-Tao; Zhao, Shui-Ping

2018-04-20

Oxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P LDL significantly increased the expression of PERK (499.5%, P LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment. This study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

Lipid Droplet Formation Is Dispensable for Endoplasmic Reticulum-associated Degradation*

Science.gov (United States)

Olzmann, James A.; Kopito, Ron R.

2011-01-01

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an “escape hatch” through which misfolded proteins, toxins, and viruses can exit the ER. We have directly tested this hypothesis by exploiting yeast strains defective in lipid droplet formation. Our data demonstrate that lipid droplet formation is dispensable for the dislocation of a plant toxin and the degradation of both soluble and integral membrane glycoproteins. PMID:21693705

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

International Nuclear Information System (INIS)

Imamura, K.; Schulz, I.

1985-01-01

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca 2+ + Mg 2+ )-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32 P from [gamma- 32 P]ATP is Ca 2+ -dependent (approximate Km for free [Ca 2+ ] = 2-3 x 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32 P incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg 2+ and monovalent cations such as K + or Na + are necessary. Vanadate inhibits both 32 P incorporation and 32 P liberation dose dependently (Km = 3 x 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32 P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca 2+ , cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca 2+ transport into rough endoplasmic reticulum from pancreatic acinar cells. The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase

GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

DEFF Research Database (Denmark)

Marzec, Michal; Eletto, Davide; Argon, Yair

2012-01-01

Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94...

Bacteria, the endoplasmic reticulum and the unfolded protein response: friends or foes?

Science.gov (United States)

Celli, Jean; Tsolis, Renée M

2015-02-01

The unfolded protein response (UPR) is a cytoprotective response that is aimed at restoring cellular homeostasis following physiological stress exerted on the endoplasmic reticulum (ER), which also invokes innate immune signalling in response to invading microorganisms. Although it has been known for some time that the UPR is modulated by various viruses, recent evidence indicates that it also has multiple roles during bacterial infections. In this Review, we describe how bacteria interact with the ER, including how bacteria induce the UPR, how subversion of the UPR promotes bacterial proliferation and how the UPR contributes to innate immune responses against invading bacteria.

Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia, Capparis, Drypetes.

Science.gov (United States)

Jørgensen, L B; Behnke, H D; Mabry, T J

1977-01-01

Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.

Mechanotransduction and Metabolism in Cardiomyocyte Microdomains.

Science.gov (United States)

Pasqualini, Francesco S; Nesmith, Alexander P; Horton, Renita E; Sheehy, Sean P; Parker, Kevin Kit

2016-01-01

Efficient contractions of the left ventricle are ensured by the continuous transfer of adenosine triphosphate (ATP) from energy production sites, the mitochondria, to energy utilization sites, such as ionic pumps and the force-generating sarcomeres. To minimize the impact of intracellular ATP trafficking, sarcomeres and mitochondria are closely packed together and in proximity with other ultrastructures involved in excitation-contraction coupling, such as t-tubules and sarcoplasmic reticulum junctions. This complex microdomain has been referred to as the intracellular energetic unit. Here, we review the literature in support of the notion that cardiac homeostasis and disease are emergent properties of the hierarchical organization of these units. Specifically, we will focus on pathological alterations of this microdomain that result in cardiac diseases through energy imbalance and posttranslational modifications of the cytoskeletal proteins involved in mechanosensing and transduction.

Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease

DEFF Research Database (Denmark)

Berchtold, M W; Brinkmeier, H; Müntener, M

2000-01-01

in the sarcoplasmic reticulum. In addition, a multitude of Ca(2+)-binding proteins is present in muscle tissue including parvalbumin, calmodulin, S100 proteins, annexins, sorcin, myosin light chains, beta-actinin, calcineurin, and calpain. These Ca(2+)-binding proteins may either exert an important role in Ca(2......Mammalian skeletal muscle shows an enormous variability in its functional features such as rate of force production, resistance to fatigue, and energy metabolism, with a wide spectrum from slow aerobic to fast anaerobic physiology. In addition, skeletal muscle exhibits high plasticity that is based...... on the potential of the muscle fibers to undergo changes of their cytoarchitecture and composition of specific muscle protein isoforms. Adaptive changes of the muscle fibers occur in response to a variety of stimuli such as, e.g., growth and differentition factors, hormones, nerve signals, or exercise...

Compartmentalization of NO signaling cascade in skeletal muscles

International Nuclear Information System (INIS)

Buchwalow, Igor B.; Minin, Evgeny A.; Samoilova, Vera E.; Boecker, Werner; Wellner, Maren; Schmitz, Wilhelm; Neumann, Joachim; Punkt, Karla

2005-01-01

Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

Science.gov (United States)

Dries, Eef; Santiago, Demetrio J; Johnson, Daniel M; Gilbert, Guillaume; Holemans, Patricia; Korte, Sanne M; Roderick, H Llewelyn; Sipido, Karin R

2016-10-15

The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca 2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through β-adrenergic receptors activates an integrated signalling cascade, enhancing Ca 2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca 2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca 2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β-adrenergic stimulation enhances Ca 2+ cycling through an integrated signalling cascade modulating L-type Ca 2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca 2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca 2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca 2+ ] i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca 2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca 2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces

PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis

Czech Academy of Sciences Publication Activity Database

Simon, S.; Skůpa, Petr; Viaene, T.; Zwiewka, M.; Tejos, R.; Klíma, Petr; Čarná, Mária; Rolčík, J.; De Rycke, R.; Moreno, I.; Dobrev, Petre; Orellana, A.; Zažímalová, Eva; Friml, J.

2016-01-01

Roč. 211, č. 1 (2016), s. 65-74 ISSN 0028-646X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GA16-10948S Institutional support: RVO:61389030 Keywords : auxin * endoplasmic reticulum (ER) * lateral root Subject RIV: ED - Physiology Impact factor: 7.330, year: 2016

Cardioprotective effect of hyperthyroidism on the stunned rat heart during ischaemia-reperfusion: energetics and role of mitochondria.

Science.gov (United States)

Ragone, María Inés; Bonazzola, Patricia; Colareda, Germán A; Consolini, Alicia E

2015-06-01

What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca(2+) handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3'-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia-reperfusion. Rats were injected s.c. daily with 20 μg kg(-1) triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca(2+) from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na(+) ] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium-calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by

Effect of metabolic syndrome and aging on Ca2+ dysfunction in coronary smooth muscle and coronary artery disease severity in Ossabaw miniature swine.

Science.gov (United States)

Badin, Jill K; Bruning, Rebecca S; Sturek, Michael

2018-05-03

Metabolic syndrome (MetS) and aging are prevalent risk factors for coronary artery disease (CAD) and contribute to the etiology of CAD, including dysregulation of Ca 2+ handling mechanisms in coronary smooth muscle (CSM). The current study tested the hypothesis that CAD severity and CSM Ca 2+ dysregulation were different in MetS-induced CAD compared to aging-induced CAD. Young (2.5 ± 0.2 years) and old (8.8 ± 1.2 years) Ossabaw miniature swine were fed an atherogenic diet for 11 months to induce MetS and were compared to lean age-matched controls. The metabolic profile was confirmed by body weight, plasma cholesterol and triglycerides, and intravenous glucose tolerance test. CAD was measured with intravascular ultrasound and histology. Intracellular Ca 2+ ([Ca 2+ ] i ) was assessed with fura-2 imaging. CAD severity was similar between MetS young and lean old swine, with MetS old swine exhibiting the most severe CAD. Compared to CSM [Ca 2+ ] i handling in lean young, the MetS young and lean old swine exhibited increased sarcoplasmic reticulum Ca 2+ store release, increased Ca 2+ influx through voltage-gated Ca 2+ channels, and attenuated sarco-endoplasmic reticulum Ca 2+ ATPase activity. MetS old and MetS young swine had similar Ca 2+ dysregulation. Ca 2+ dysregulation, mainly the SR Ca 2+ store, in CSM is more pronounced in lean old swine, which is indicative of mild, proliferative CAD. MetS old and MetS young swine exhibit Ca 2+ dysfunction that is typical of late, severe disease. The more advanced, complex plaques in MetS old swine suggest that the "aging milieu" potentiates effects of Ca 2+ handling dysfunction in CAD. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of the low- versus high-intensity exercise training on endoplasmic reticulum stress and GLP-1 in adolescents with type 2 diabetes mellitus.

Science.gov (United States)

Lee, Sung Soo; Yoo, Jae Ho; So, Yong Seok

2015-10-01

[Purpose] The primary objective of this study was to investigate the effect of low-intensity exercise training compare with high-intensity exercise training on endoplasmic reticulum stress and glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus. [Subjects and Methods] The low-intensity exercise training group performed aerobic exercise training at an intensity of ≤ 45% of the heart rate reserve. The high-intensity interval exercise training group performed interval exercise training at an intensity of ≥ 80% of the heart rate reserve. The exercise-related energy consumption was determined for both groups on a per-week basis (1,200 kcal/week). [Results] Both groups showed improvement in the glucose-regulated protein 78 and dipeptidyl peptidase-4, but the size of the between-group effect was not statistically significant. The high-intensity interval exercise training group showed a significant reduction in percentage body fat. The C-peptide level increased after the 12-weeks programs and was significantly different, between the groups. Fasting glucose, insulin resistance in the fasting state according to homeostasis model assessment, and leptin decreased after the 12-weeks exercise program and were significantly different between the groups, and glucagon-like peptide-1 increased after the 12-week exercise programs and was significantly different between the groups. [Conclusion] In conclusion high-intensity interval exercise training, as defined in this study, may lead to improvements in body composition, glycemic control, endoplasmic reticulum stress, and the glucagon-like peptide-1 in adolescents with type 2 diabetes mellitus.

p53-inducible DHRS3 Is an Endoplasmic Reticulum Protein Associated with Lipid Droplet Accumulation*

Science.gov (United States)

Deisenroth, Chad; Itahana, Yoko; Tollini, Laura; Jin, Aiwen; Zhang, Yanping

2011-01-01

The transcription factor p53 plays a critical role in maintaining homeostasis as it relates to cellular growth, proliferation, and metabolism. In an effort to identify novel p53 target genes, a microarray approach was utilized to identify DHRS3 (also known as retSDR1) as a robust candidate gene. DHRS3 is a highly conserved member of the short chain alcohol dehydrogenase/reductase superfamily with a reported role in lipid and retinoid metabolism. Here, we demonstrate that DHRS3 is an endoplasmic reticulum (ER) protein that is shuttled to the ER via an N-terminal endoplasmic reticulum targeting signal. One important function of the ER is synthesis of neutral lipids that are packaged into lipid droplets whose biogenesis occurs from ER-derived membranes. DHRS3 is enriched at focal points of lipid droplet budding where it also localizes to the phospholipid monolayer of ER-derived lipid droplets. p53 promotes lipid droplet accumulation in a manner consistent with DHRS3 enrichment in the ER. As a p53 target gene, the observations of Dhrs3 location and potential function provide novel insight into an unexpected role for p53 in lipid droplet dynamics with implications in cancer cell metabolism and obesity. PMID:21659514

Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Xin; Xu, Mei; Frank, Jacqueline A. [Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536 (United States); Ke, Zun-ji [Department of Biochemistry, Shanghai University of Traditional Chinese Medicine, Shanghai, China 201203 (China); Luo, Jia, E-mail: jialuo888@uky.edu [Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536 (United States); Department of Biochemistry, Shanghai University of Traditional Chinese Medicine, Shanghai, China 201203 (China)

2017-04-01

Thiamine (vitamin B1) deficiency (TD) plays a major role in the etiology of Wernicke's encephalopathy (WE) which is a severe neurological disorder. TD induces selective neuronal cell death, neuroinflammation, endoplasmic reticulum (ER) stress and oxidative stress in the brain which are commonly observed in many aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and progressive supranuclear palsy (PSP). However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research is hindered due to the lack of appropriate in vitro models. The neurons derived for the human induced pluripotent stem cells (hiPSCs) provide a relevant and powerful tool for the research in pharmaceutical and environmental neurotoxicity. In this study, we for the first time used human induced pluripotent stem cells (hiPSCs)-derived neurons (iCell neurons) to investigate the mechanisms of TD-induced neurodegeneration. We showed that TD caused a concentration- and duration-dependent death of iCell neurons. TD induced ER stress which was evident by the increase in ER stress markers, such as GRP78, XBP-1, CHOP, ATF-6, phosphorylated eIF2α, and cleaved caspase-12. TD also triggered oxidative stress which was shown by the increase in the expression 2,4-dinitrophenyl (DNP) and 4-hydroxynonenal (HNE). ER stress inhibitors (STF-083010 and salubrinal) and antioxidant N-acetyl cysteine (NAC) were effective in alleviating TD-induced death of iCell neurons, supporting the involvement of ER stress and oxidative stress. It establishes that the iCell neurons are a novel tool to investigate cellular and molecular mechanisms for TD-induced neurodegeneration. - Highlights: • Thiamine deficiency (TD) causes death of human neurons in culture. • TD induces both endoplasmic reticulum (ER) stress and oxidative stress. • Alleviating ER stress and oxidative stress reduces TD

Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells

International Nuclear Information System (INIS)

Wang, Xin; Xu, Mei; Frank, Jacqueline A.; Ke, Zun-ji; Luo, Jia

2017-01-01

Thiamine (vitamin B1) deficiency (TD) plays a major role in the etiology of Wernicke's encephalopathy (WE) which is a severe neurological disorder. TD induces selective neuronal cell death, neuroinflammation, endoplasmic reticulum (ER) stress and oxidative stress in the brain which are commonly observed in many aging-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and progressive supranuclear palsy (PSP). However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research is hindered due to the lack of appropriate in vitro models. The neurons derived for the human induced pluripotent stem cells (hiPSCs) provide a relevant and powerful tool for the research in pharmaceutical and environmental neurotoxicity. In this study, we for the first time used human induced pluripotent stem cells (hiPSCs)-derived neurons (iCell neurons) to investigate the mechanisms of TD-induced neurodegeneration. We showed that TD caused a concentration- and duration-dependent death of iCell neurons. TD induced ER stress which was evident by the increase in ER stress markers, such as GRP78, XBP-1, CHOP, ATF-6, phosphorylated eIF2α, and cleaved caspase-12. TD also triggered oxidative stress which was shown by the increase in the expression 2,4-dinitrophenyl (DNP) and 4-hydroxynonenal (HNE). ER stress inhibitors (STF-083010 and salubrinal) and antioxidant N-acetyl cysteine (NAC) were effective in alleviating TD-induced death of iCell neurons, supporting the involvement of ER stress and oxidative stress. It establishes that the iCell neurons are a novel tool to investigate cellular and molecular mechanisms for TD-induced neurodegeneration. - Highlights: • Thiamine deficiency (TD) causes death of human neurons in culture. • TD induces both endoplasmic reticulum (ER) stress and oxidative stress. • Alleviating ER stress and oxidative stress reduces TD

Taurine Rescues Cisplatin-Induced Muscle Atrophy In Vitro: A Morphological Study

Directory of Open Access Journals (Sweden)

Alessandra Stacchiotti

2014-01-01

Full Text Available Cisplatin (CisPt is a widely used chemotherapeutic drug whose side effects include muscle weakness and cachexia. Here we analysed CisPt-induced atrophy in C2C12 myotubes by a multidisciplinary morphological approach, focusing on the onset and progression of autophagy, a protective cellular process that, when excessively activated, may trigger protein hypercatabolism and atrophy in skeletal muscle. To visualize autophagy we used confocal and transmission electron microscopy at different times of treatment and doses of CisPt. Moreover we evaluated the effects of taurine, a cytoprotective beta-amino acid able to counteract oxidative stress, apoptosis, and endoplasmic reticulum stress in different tissues and organs. Our microscopic results indicate that autophagy occurs very early in 50 μM CisPt challenged myotubes (4 h–8 h before overt atrophy but it persists even at 24 h, when several autophagic vesicles, damaged mitochondria, and sarcoplasmic blebbings engulf the sarcoplasm. Differently, 25 mM taurine pretreatment rescues the majority of myotubes size upon 50 μM CisPt at 24 h. Taurine appears to counteract atrophy by restoring regular microtubular apparatus and mitochondria and reducing the overload and the localization of autophagolysosomes. Such a promising taurine action in preventing atrophy needs further molecular and biochemical studies to best define its impact on muscle homeostasis and the maintenance of an adequate skeletal mass in vivo.

Plant transducers of the endoplasmic reticulum unfolded protein response

KAUST Repository

Iwata, Yuji; Koizumi, Nozomu

2012-01-01

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.

Plant transducers of the endoplasmic reticulum unfolded protein response

KAUST Repository

Iwata, Yuji

2012-12-01

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.

Fluoride-elicited developmental testicular toxicity in rats: Roles of endoplasmic reticulum stress and inflammatory response

Energy Technology Data Exchange (ETDEWEB)

Zhang, Shun [Department of Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, Hubei (China); Jiang, Chunyang [Department of Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, Hubei (China); Department of Thoracic Surgery, Tianjin Union Medicine Centre, 190 Jieyuan Road, Hongqiao District, Tianjin 300121, Tianjin (China); Liu, Hongliang [Tianjin Center for Disease Control and Prevention, Huayue Road 6, Hedong Region, Tianjin 300011, Tianjin (China); Guan, Zhizhong [Department of Pathology, Guiyang Medical College, Guiyang 550004, Guizhou (China); Zeng, Qiang [Tianjin Center for Disease Control and Prevention, Huayue Road 6, Hedong Region, Tianjin 300011, Tianjin (China); Zhang, Cheng; Lei, Rongrong; Xia, Tao; Gao, Hui; Yang, Lu; Chen, Yihu; Wu, Xue; Zhang, Xiaofei [Department of Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, Hubei (China); Cui, Yushan; Yu, Linyu [Tianjin Center for Disease Control and Prevention, Huayue Road 6, Hedong Region, Tianjin 300011, Tianjin (China); Wang, Zhenglun [Department of Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, Hubei (China); Wang, Aiguo, E-mail: wangaiguo@mails.tjmu.edu.cn [Department of Environmental Health and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, Hubei (China)

2013-09-01

Long-term excessive fluoride intake is known to be toxic and can damage a variety of organs and tissues in the human body. However, the molecular mechanisms underlying fluoride-induced male reproductive toxicity are not well understood. In this study, we used a rat model to simulate the situations of human exposure and aimed to evaluate the roles of endoplasmic reticulum (ER) stress and inflammatory response in fluoride-induced testicular injury. Sprague–Dawley rats were administered with sodium fluoride (NaF) at 25, 50 and 100 mg/L via drinking water from pre-pregnancy to gestation, birth and finally to post-puberty. And then the testes of male offspring were studied at 8 weeks of age. Our results demonstrated that fluoride treatment increased MDA accumulation, decreased SOD activity, and enhanced germ cell apoptosis. In addition, fluoride elevated mRNA and protein levels of glucose-regulated protein 78 (GRP78), inositol requiring ER-to-nucleus signal kinase 1 (IRE1), and C/EBP homologous protein (CHOP), indicating activation of ER stress signaling. Furthermore, fluoride also induced testicular inflammation, as manifested by gene up-regulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in a nuclear factor-κB (NF-κB)-dependent manner. These were associated with marked histopathological lesions including injury of spermatogonia, decrease of spermatocytes and absence of elongated spermatids, as well as severe ultrastructural abnormalities in testes. Taken together, our results provide compelling evidence that ER stress and inflammation would be novel and significant mechanisms responsible for fluoride-induced disturbance of spermatogenesis and germ cell loss in addition to oxidative stress. - Highlights: • We used a rat model to simulate the situations of human fluoride (F) exposure. • Developmental F exposure induces testicular damage related with oxidative stress. �

Fluoride-elicited developmental testicular toxicity in rats: Roles of endoplasmic reticulum stress and inflammatory response

International Nuclear Information System (INIS)

Zhang, Shun; Jiang, Chunyang; Liu, Hongliang; Guan, Zhizhong; Zeng, Qiang; Zhang, Cheng; Lei, Rongrong; Xia, Tao; Gao, Hui; Yang, Lu; Chen, Yihu; Wu, Xue; Zhang, Xiaofei; Cui, Yushan; Yu, Linyu; Wang, Zhenglun; Wang, Aiguo

2013-01-01

Long-term excessive fluoride intake is known to be toxic and can damage a variety of organs and tissues in the human body. However, the molecular mechanisms underlying fluoride-induced male reproductive toxicity are not well understood. In this study, we used a rat model to simulate the situations of human exposure and aimed to evaluate the roles of endoplasmic reticulum (ER) stress and inflammatory response in fluoride-induced testicular injury. Sprague–Dawley rats were administered with sodium fluoride (NaF) at 25, 50 and 100 mg/L via drinking water from pre-pregnancy to gestation, birth and finally to post-puberty. And then the testes of male offspring were studied at 8 weeks of age. Our results demonstrated that fluoride treatment increased MDA accumulation, decreased SOD activity, and enhanced germ cell apoptosis. In addition, fluoride elevated mRNA and protein levels of glucose-regulated protein 78 (GRP78), inositol requiring ER-to-nucleus signal kinase 1 (IRE1), and C/EBP homologous protein (CHOP), indicating activation of ER stress signaling. Furthermore, fluoride also induced testicular inflammation, as manifested by gene up-regulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in a nuclear factor-κB (NF-κB)-dependent manner. These were associated with marked histopathological lesions including injury of spermatogonia, decrease of spermatocytes and absence of elongated spermatids, as well as severe ultrastructural abnormalities in testes. Taken together, our results provide compelling evidence that ER stress and inflammation would be novel and significant mechanisms responsible for fluoride-induced disturbance of spermatogenesis and germ cell loss in addition to oxidative stress. - Highlights: • We used a rat model to simulate the situations of human fluoride (F) exposure. • Developmental F exposure induces testicular damage related with oxidative stress. �

Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

Science.gov (United States)

Wei, Shun-Guang; Yu, Yang; Weiss, Robert M; Felder, Robert B

2016-10-01

We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 μg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.

Mechanisms of Alcohol-Induced Endoplasmic Reticulum Stress and Organ Injuries

Directory of Open Access Journals (Sweden)

Cheng Ji

2012-01-01

Full Text Available Alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, which affect virtually all biological processes inside the cell. Excessive alcohol consumption induces numerous pathological stress responses, part of which is endoplasmic reticulum (ER stress response. ER stress, a condition under which unfolded/misfolded protein accumulates in the ER, contributes to alcoholic disorders of major organs such as liver, pancreas, heart, and brain. Potential mechanisms that trigger the alcoholic ER stress response are directly or indirectly related to alcohol metabolism, which includes toxic acetaldehyde and homocysteine, oxidative stress, perturbations of calcium or iron homeostasis, alterations of S-adenosylmethionine to S-adenosylhomocysteine ratio, and abnormal epigenetic modifications. Interruption of the ER stress triggers is anticipated to have therapeutic benefits for alcoholic disorders.

Changes in amino transferases and muscle proteins when treating pigmeat with ionising rays

International Nuclear Information System (INIS)

Hamm, R.; Hofmann, K.; Gruenewald, T.; Partmann, W.

1975-01-01

Slices of lean pigmeat were treated with electron beams doses of 0.2, 1.0 and 5.0 Mrad. Low irradiation doses led to an increase in the activity of aspartate amino transferase (GOT) and alanin amino transferase (GPT) in the tissue generally and in the sarcoplasm (juice expressed from the muscle). 5 Mrad caused a great reduction in the activity of GOT and GPT in the tissue and the sarcoplasm. It seems doubtful whether this inactivation is due to a destruction of enzyme sulphhydryl groups. Irradiating with 5 Mrad resulted in partial release of the mitochondrial GOT isozyme (GOTsub(M)) into the sarcoplasm. This indicates damage to the mitochondrial membranes by ionising radiation. Irradiating the pigmeat increased the pH of the tissue and lowered its water binding ability (increase in drip). Up to a dose of 1 Mrad the solubility of the sarcoplasmic proteins was not definitely affected, but 5 Mrad caused a considerable drop in protein solubility. Surprisingly a dose of even 5 Mrad did not change the total number of sulphhydryl groups present in the tissue. Sephadex thin layer electrophoresis showed that at 0.2 Mrad there was a drastic decrease in the myosin band and an increase in peptide fragments of low molecular weight, whilst actin was little changed. (orig.) [de

Endoplasmic reticulum stress regulates inflammation and insulin resistance in skeletal muscle from pregnant women.

Science.gov (United States)

Liong, Stella; Lappas, Martha

2016-04-15

Sterile inflammation and infection are key mediators of inflammation and peripheral insulin resistance associated with gestational diabetes mellitus (GDM). Studies have shown endoplasmic reticulum (ER) stress to induce inflammation and insulin resistance associated with obesity and type 2 diabetes, however is paucity of studies investigating the effects of ER stress in skeletal muscle on inflammation and insulin resistance associated with GDM. ER stress proteins IRE1α, GRP78 and XBP-1s were upregulated in skeletal muscle of obese pregnant women, whereas IRE1α was increased in GDM women. Suppression of ER stress, using ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or siRNA knockdown of IRE1α and GRP78, significantly downregulated LPS-, poly(I:C)- or IL-1β-induced production of IL-6, IL-8, IL-1β and MCP-1. Furthermore, LPS-, poly(I:C)- or TNF-α-induced insulin resistance was improved following suppression of ER stress, by increasing insulin-stimulated phosphorylation of IR-β, IRS-1, GLUT-4 expression and glucose uptake. In summary, our inducible obesity and GDM-like models suggests that the development of GDM may be involved in activating ER stress-induced inflammation and insulin resistance in human skeletal muscle. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Coupling of the Functional Stability of Rat Myocardium and Activity of Lipid Peroxidation in Combined Development of Postinfarction Remodeling and Diabetes Mellitus

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S. A. Afanasiev

2016-01-01

Full Text Available Coupling of the functional stability of rat myocardium and activity of lipid peroxidation processes in combined development of postinfarction remodeling and diabetes mellitus has been studied. The functional stability of myocardium was studied by means of the analysis of inotropic reaction on extrasystolic stimulus, the degree of left ventricular hypertrophy, and the size of scar zone. It was shown that in combined development of postinfarction cardiac remodeling of heart (PICR with diabetes mellitus (DM animal body weight decreased in less degree than in diabetic rats. Animals with combined pathology had no heart hypertrophy. The amplitude of extrasystolic contractions in rats with PICR combined with DM had no differences compared to the control group. In myocardium of rats with PICR combined with DM postextrasystolic potentiation was observed in contrast with the rats with PICR alone. The rats with combined pathology had the decreased value of TBA-active products. Thus, the results of study showed that induction of DM on the stage of the development of postinfarction remodeling increases adaptive ability of myocardium. It is manifested in inhibition of increase of LPO processes activity and maintaining of force-interval reactions of myocardium connected with calcium transport systems of sarcoplasmic reticulum of cardiomyocytes.

Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

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Jie Yang

2016-11-01

Full Text Available Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A, a regulatory subunit of protein phosphatase 2A (PP2A, is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR, plays an important role in the excitation-contraction (EC coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated proteins (Cas system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening. Hematoxylin and eosin (H&E staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT. Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

Effect of ascorbic acid on fatigue of skeletal muscle fibres in long term cold exposed sprague dawley rats

International Nuclear Information System (INIS)

Rashid, A.; Ayub, M.

2011-01-01

On exposure to prolonged cold temperature, the body responds for effective heat production both by shivering and non-shivering thermo genesis. Cold exposure increases the production of reactive oxygen species which influence the sarcoplasmic reticulum Ca/sup ++/ release from the skeletal muscles and affect their contractile properties. The role of ascorbic acid supplementation on force of contraction during fatigue of cold exposed skeletal muscles was evaluated in this study. Method: Ninety healthy, male Sprague Dawley rats were randomly divided into three groups of control, cold exposed, and cold exposed with ascorbic acid 500 mg/L supplementation mixed in drinking water. Group II and III were given cold exposure by keeping their cages in ice-filled tubs for 1 hr/day for one month. After one month, the extensor digitorum longus muscle was dissected out and force of contraction during fatigue in the skeletal muscle fibres was analysed on a computerised data acquisition system. Results: The cold exposed group showed a significant delay in the force of contraction during fatigue of skeletal muscle fibres compared to control group. Group III showed easy fatigability and a better force of contraction than the cold exposed group. Conclusions: Ascorbic acid increases the force of contraction and decreases resistance to fatigue in the muscles exposed to chronic cold. (author)

Aerobic Training Prevents Heatstrokes in Calsequestrin-1 Knockout Mice by Reducing Oxidative Stress

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Flávia Alessandra Guarnier

2018-01-01

Full Text Available Calsequestrin-1 knockout (CASQ1-null mice suffer lethal episodes when exposed to strenuous exercise and environmental heat, crises known as exertional/environmental heatstroke (EHS. We previously demonstrated that administration of exogenous antioxidants (N-acetylcysteine and trolox reduces CASQ1-null mortality during exposure to heat. As aerobic training is known to boost endogenous antioxidant protection, we subjected CASQ1-null mice to treadmill running for 2 months at 60% of their maximal speed for 1 h, 5 times/week. When exposed to heat stress protocol (41°C/1 h, the mortality rate of CASQ1-null mice was significantly reduced compared to untrained animals (86% versus 16%. Protection from heatstrokes was accompanied by a reduced increase in core temperature during the stress protocol and by an increased threshold of response to caffeine of isolated extensor digitorum longus muscles during in vitro contracture test. At cellular and molecular levels, aerobic training (i improved mitochondrial function while reducing their damage and (ii lowered calpain activity and lipid peroxidation in membranes isolated from sarcoplasmic reticulum and mitochondria. Based on this evidence, we hypothesize that the protective effect of aerobic training is essentially mediated by a reduction in oxidative stress during exposure of CASQ1-null mice to adverse environmental conditions.

Curcumin inhibits endoplasmic reticulum stress induced by cerebral ischemia-reperfusion injury in rats

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Zhu, Haiying; Fan, Yanxia; Sun, Hongyu; Chen, Liyan; Man, Xiao

2017-01-01

The aim of the present study was to observe the dynamic changes of the growth arrest and DNA damage-inducible 153 (GADD153) gene and caspase-12 in the brain tissue of rats with cerebral ischemia-reperfusion injury (CIRI) and the impact of curcumin pretreatment. A total of 60 rats were randomly divided into the normal group (N), the sham operation group (S), the dimethyl sulfoxide control group (D) and the curcumin treatment group (C). For group D and C, 12 (T1), 24 (T2) and 72 h (T3) of reperfusion were performed after 2 h ischemia. The expression levels of GADD153 and caspase-12 in the brain tissue were detected and compared among the groups by immunohistochemistry, immunofluorescence double staining and western blotting. The expression levels of GADD153 and caspase-12 were increased at T1compared with groups N and S, and the expression of caspase-12 peaked at T2 in group D, while GADD153 was increased until T3 in group D. Compared with group D, the expression levels of GADD153 and caspase-12 in group C at T2 and T3 were significantly decreased (P<0.05). Endoplasmic reticulum stress is involved in the pathological process of CIRI. Curcumin may decrease the expression levels of the above two factors, thus exhibiting protective effects against CIRI in rats. PMID:29067098

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney.

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De Miguel, Carmen; Hamrick, William C; Hobbs, Janet L; Pollock, David M; Carmines, Pamela K; Pollock, Jennifer S

2017-02-23

Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ET B deficient (ET B def) or transgenic control (TG-con) rats were used in the presence or absence of ET A receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ET B def rats showed a 14-24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ET A blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ET B def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ET A receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ET B receptor has protective effects. These results highlight targeting the ET A receptor as a therapeutic approach against ER stress-induced kidney injury.

Kaempferol induces hepatocellular carcinoma cell death via endoplasmic reticulum stress-CHOP-autophagy signaling pathway.

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Guo, Haiqing; Lin, Wei; Zhang, Xiangying; Zhang, Xiaohui; Hu, Zhongjie; Li, Liying; Duan, Zhongping; Zhang, Jing; Ren, Feng

2017-10-10

Kaempferol is a flavonoid compound that has gained widespread attention due to its antitumor functions. However, the underlying mechanisms are still not clear. The present study investigated the effect of kaempferol on hepatocellular carcinoma and its underlying mechanisms. Kaempferol induced autophagy in a concentration- and time-dependent manner in HepG2 or Huh7 cells, which was evidenced by the significant increase of autophagy-related genes. Inhibition of autophagy pathway, through 3-methyladenine or Atg7 siRNA, strongly diminished kaempferol-induced apoptosis. We further hypothesized that kaempferol can induce autophagy via endoplasmic reticulum (ER) stress pathway. Indeed, blocking ER stress by 4-phenyl butyric acid (4-PBA) or knockdown of CCAAT/enhancer-binding protein homologous protein (CHOP) with siRNA alleviated kaempferol-induced HepG2 or Huh7 cells autophagy; while transfection with plasmid overexpressing CHOP reversed the effect of 4-PBA on kaempferol-induced autophagy. Our results demonstrated that kaempferol induced hepatocarcinoma cell death via ER stress and CHOP-autophagy signaling pathway; kaempferol may be used as a potential chemopreventive agent for patients with hepatocellular carcinoma.

Radioresistant Spodoptera frugiperda 9 insect cells display excessive resistance to 'endoplasmic reticulum' stress and calcium disturbances via pre-emptive activation of unfolded protein response pathway

International Nuclear Information System (INIS)

Guleria, Ayushi; Chandna, Sudhir

2016-01-01

Endoplasmic Reticulum (ER) performs multiple cellular functions such as proper folding of newly synthesized proteins and calcium homeostasis. ER stress triggers unfolded protein response (UPR) that attempts to restore normal ER function and resists damage-induced cell death. Lepidopteran Sf9 insect cells (derived from Spodoptera frugiperda) display 100-200 times higher radioresistance than mammalian cells. We have earlier reported that gamma-radiation doses <1000 Gy fail to trigger increase in cytosolic calcium in Sf9 cells, indicating resilience to calcium/ ER disturbances

Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU

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Francesco Chemello

2015-09-01

Full Text Available The mitochondrial calcium uniporter (MCU gene codifies for the inner mitochondrial membrane (IMM channel responsible for mitochondrial Ca2+ uptake. Cytosolic Ca2+ transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2+ regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2+ transients elicit large increases in the [Ca2+] of the mitochondrial matrix ([Ca2+]mt. Mitochondrial Ca2+ uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2+ uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2+ uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection. Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/ (GSE60931.

Coexistence of potentiation and fatigue in skeletal muscle

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D.E. Rassier

2000-05-01

Full Text Available Twitch potentiation and fatigue in skeletal muscle are two conditions in which force production is affected by the stimulation history. Twitch potentiation is the increase in the twitch active force observed after a tetanic contraction or during and following low-frequency stimulation. There is evidence that the mechanism responsible for potentiation is phosphorylation of the regulatory light chains of myosin, a Ca2+-dependent process. Fatigue is the force decrease observed after a period of repeated muscle stimulation. Fatigue has also been associated with a Ca2+-related mechanism: decreased peak Ca2+ concentration in the myoplasm is observed during fatigue. This decrease is probably due to an inhibition of Ca2+ release from the sarcoplasmic reticulum. Although potentiation and fatigue have opposing effects on force production in skeletal muscle, these two presumed mechanisms can coexist. When peak myoplasmic Ca2+ concentration is depressed, but myosin light chains are relatively phosphorylated, the force response can be attenuated, not different, or enhanced, relative to previous values. In circumstances where there is interaction between potentiation and fatigue, care must be taken in interpreting the contractile responses.

Numerical Analysis of the Effect of T-tubule Location on Calcium Transient in Ventricular Myocytes

Science.gov (United States)

George, Uduak Z.; Wang, Jun; Yu, Zeyun

2013-01-01

Intracellular calcium (Ca2+) signaling in cardiac myocytes is vital for proper functioning of the heart. Understanding the intracellular Ca2+ dynamics would give an insight into the functions of normal and diseased hearts. In the current study, spatiotemporal Ca2+ dynamics is investigated in ventricular myocytes by considering Ca2+ release and re-uptake via sarcolemma and transverse tubules (T-tubules), Ca2+ diffusion and buffering in the cytosol, and the blockade of Ca2+ activities associated with the sarcoplasmic reticulum. This study is carried out using a three dimensional (3D) geometric model of a branch of T-tubule extracted from the electron microscopy (EM) images of a partial ventricular myocyte. Mathematical modeling is done by using a system of partial differential equations involving Ca2+ , buffers, and membrane channels. Numerical simulation results suggest that a lack of T-tubule structure at the vicinity of the cell surface could increase the peak time of Ca2+ concentration in myocytes. The results also show that T-tubules and mobile buffers play an important role in the regulation of Ca2+ transient in ventricular myocytes. PMID:24212025

Hypertensive effects of the iv administration of picomoles of ouabain

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A.S. Padilha

2011-09-01

Full Text Available Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na+ pump, inhibiting its activity. Inhibition of this pump increases intracellular Na+, which reduces the activity of the sarcolemmal Na+/Ca2+ exchanger and thereby reduces Ca2+ extrusion. Consequently, intracellular Ca2+ increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.

A Molecular Web: Endoplasmic Reticulum Stress, Inflammation and Oxidative Stress

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Namrata eChaudhari

2014-07-01

Full Text Available Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded protein response (UPR through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS. Toxic accumulation of ROS within ER and mitochondria disturb fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways has been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease and others. In this review we have discussed the UPR signaling pathways, and networking between ER stress induced inflammatory pathways, oxidative stress and mitochondrial signaling events which further induce or exacerbate ER stress.

Association of the golgi UDP-galactose transporter with UDP-galactose: ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

NARCIS (Netherlands)

Sprong, H.; Degroote, S.; Nilsson, T.; Kawakita, M.; Ishida, N.; van der Sluijs, P.; van Meer, G.

2003-01-01

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose: ceramide galactosyltransferase. It is not known how

Psychological Stress, Cocaine and Natural Reward Each Induce Endoplasmic Reticulum Stress Genes in Rat Brain

OpenAIRE

Pavlovsky, Ashly A.; Boehning, Darren; Li, Dingge; Zhang, Yafang; Fan, Xiuzhen; Green, Thomas A.

2013-01-01

Our prior research has shown that the transcription of endoplasmic reticulum (ER) stress transcription factors Activating Transcription Factor 3 (ATF3) and ATF4 are induced by amphetamine and restraint stress in rat striatum. However, presently it is unknown the full extent of ER stress responses to psychological stress or cocaine, and which of the three ER stress pathways is activated. The current study examines transcriptional responses of key ER stress target genes subsequent to psychologi...

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

OpenAIRE

Goto, Asako; Charman, Mark; Ridgway, Neale D.

2015-01-01

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to de...

STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

Science.gov (United States)

Carrasco, Silvia; Meyer, Tobias

2011-01-01

Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

Endoplasmic Reticulum Stress-Related Factors Protect against Diabetic Retinopathy

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Wei-Kun Hu

2012-01-01

Full Text Available The endoplasmic reticulum (ER is a principal mediator of signal transduction in the cell, and disruption of its normal function (a mechanism known as ER stress has been associated with the pathogenesis of several diseases. ER stress has been demonstrated to contribute to onset and progression of diabetic retinopathy (DR by induction of multiple inflammatory signaling pathways. Recent studies have begun to describe the gene expression profile of ER stress-related genes in DR; moreover, genes that play a protective role against DR have been identified. P58IPK was determined to be able to reduce retinal vascular leakage under high glucose conditions, thus protecting retinal cells. It has also been found by our lab that ER-associated protein degradation factors exhibit significantly different expression patterns in rat retinas under sustained high glucose conditions. Future research based upon these collective genomic findings will contribute to our overall understanding of DR pathogenesis as well as identify potential therapeutic targets.

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

DEFF Research Database (Denmark)

Thastrup, Ole; Cullen, P J; Drøbak, B K

1990-01-01

. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum...

Endoplasmic reticulum stress is induced in the human placenta during labour.

Science.gov (United States)

Veerbeek, J H W; Tissot Van Patot, M C; Burton, G J; Yung, H W

2015-01-01

Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tang-Luo-Ning, a Traditional Chinese Medicine, Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis of Schwann Cells under High Glucose Environment

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Weijie Yao

2017-01-01

Full Text Available Tang-Luo-Ning (TLN has a definite effect in the clinical treatment of diabetic peripheral neuropathy (DPN. Schwann cells (SCs apoptosis induced by endoplasmic reticulum stress (ER stress is one of the main pathogeneses of DPN. This study investigates whether TLN can inhibit SCs apoptosis by inhibiting ER stress-induced apoptosis. Our previous researches have demonstrated that TLN could increase the expression of ER stress marker protein GRP78 and inhibited the expression of apoptosis marker protein CHOP in ER stress. In this study, the results showed that TLN attenuated apoptosis by decreasing Ca2+ level in SCs and maintaining ER morphology. TLN could decrease downstream proteins of CHOP including GADD34 and Ero1α, while it increased P-eIF2α and decreased the upstream proteins of CHOP including P-IRE1α/IRE1α and XBP-1, thereby reducing ER stress-induced apoptosis.

Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain.

Science.gov (United States)

Kreiling, Jill A; Balantac, Zaneta L; Crawford, Andrew R; Ren, Yuexin; Toure, Jamal; Zchut, Sigalit; Kochilas, Lazaros; Creton, Robbert

2008-01-01

Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.

Cyclosporine A-sensitive, cyclophilin B-dependent endoplasmic reticulum-associated degradation.

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Riccardo Bernasconi

2010-09-01

Full Text Available Peptidyl-prolyl cis/trans isomerases (PPIs catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-L(S substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells.

Cyclosporine A-Sensitive, Cyclophilin B-Dependent Endoplasmic Reticulum-Associated Degradation

Science.gov (United States)

Luban, Jeremy; Molinari, Maurizio

2010-01-01

Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells. PMID:20927389

Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

DEFF Research Database (Denmark)

Laursen, Mette; Bublitz, Maike; Moncoq, Karine

2009-01-01

is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

Pekinenin E Inhibits the Growth of Hepatocellular Carcinoma by Promoting Endoplasmic Reticulum Stress Mediated Cell Death

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Lu Fan

2017-06-01

Full Text Available Hepatocellular carcinoma (HCC is a malignant primary liver cancer with poor prognosis. In the present study, we report that pekinenin E (PE, a casbane diterpenoid derived from the roots of Euphorbia pekinensis, has a strong antitumor activity against human HCC cells both in vitro and in vivo. PE suppressed the growth of human HCC cells Hep G2 and SMMC-7721. In addition, PE-mediated endoplasmic reticulum (ER stress caused increasing expressions of C/EBP homologous protein (CHOP, leading to apoptosis in HCC cells both in vitro and in vivo. Inhibition of ER stress with CHOP small interfering RNA or 4-phenyl-butyric acid partially reversed PE-induced cell death. Furthermore, PE induced S cell cycle arrest, which could also be partially reversed by CHOP knockdown. In all, these findings suggest that PE causes ER stress-associated cell death and cell cycle arrest, and it may serve as a potent agent for curing human HCC.

Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release.

Science.gov (United States)

van Kuppeveld, F J; Hoenderop, J G; Smeets, R L; Willems, P H; Dijkman, H B; Galama, J M; Melchers, W J

1997-01-01

Digital-imaging microscopy was performed to study the effect of Coxsackie B3 virus infection on the cytosolic free Ca2+ concentration and the Ca2+ content of the endoplasmic reticulum (ER). During the course of infection a gradual increase in the cytosolic free Ca2+ concentration was observed, due to the influx of extracellular Ca2+. The Ca2+ content of the ER decreased in time with kinetics inversely proportional to those of viral protein synthesis. Individual expression of protein 2B was sufficient to induce the influx of extracellular Ca2+ and to release Ca2+ from ER stores. Analysis of mutant 2B proteins showed that both a cationic amphipathic alpha-helix and a second hydrophobic domain in 2B were required for these activities. Consistent with a presumed ability of protein 2B to increase membrane permeability, viruses carrying a mutant 2B protein exhibited a defect in virus release. We propose that 2B gradually enhances membrane permeability, thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny. PMID:9218794

The effect of metformin treatment on endoplasmic reticulum (ER stress induced by status epilepticus (SE via the PERK-eIF2α-CHOP pathway

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Jing Chen

2018-02-01

Full Text Available Status epilepticus (SE is defined as continuous seizure activity lasting more than 5 minutes. It results in neuronal cell death, mediated by endoplasmic reticulum (ER stress response. Previously, metformin demonstrated neuroprotective effects in primary cortical neurons. In this study, we analyzed the effect of metformin on ER stress via the pro-apoptotic protein kinase RNA-like endoplasmic reticulum kinase (PERK-eukaryotic initiation factor 2α (eIF2α-C/EBP homologous protein (CHOP pathway. SE was induced in rats by pentylenetetrazole. Following SE, the rats were treated with salubrinal, GSK2656157, or metformin. In a control group (normal saline SE was not induced. CHOP, eIF2α, and PERK expression was determined by Western blot; apoptosis was analyzed by TUNEL assay. CHOP expression was significantly increased at 6 and 24 hours following SE. At both time points, eIF2α and PERK levels were also increased. At 6 hours, CHOP expression was significantly reduced in salubrinal, GSK2656157 and metformin groups versus SE group. eIF2α and PERK levels were decreased in metformin compared to SE group. eIF2α expression was markedly decreased in salubrinal versus SE group, while PERK expression was markedly reduced in GSK2656157 versus SE group. At 6 and 24 hours, the apoptosis rate was significantly increased in SE versus control group, while it was significantly reduced in salubrinal, GSK2656157, and metformin groups compared to SE group. The apoptosis rate also decreased in salubrinal group at 24 hours, although not to the extent observed in metformin group. Overall, CHOP expression and apoptosis induced by SE in rats were reduced with metformin. Further studies are required to evaluate the clinical relevance of metformin for patients with SE.

[Activation of endoplasmic reticulum stress and its effect on osteogenic differentiation induced by micropit/nanotube topography].

Science.gov (United States)

Shi, M Q; Song, W; Han, T X; Chang, B; Zhang, Y M

2017-02-09

Objective: To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropit/nanotube topography (MNT), so as to provide guidance for the topography design of biomaterials. Methods: Four sample groups were fabricated: polishing control group (polished titanium, PT, no treatment), thapsigargin treatment (TG, 0.1 μmol/L TG treated for 9 h), MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching). Scanning electron microscope (SEM) was used to observe the topography of Ti samples. The alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7, 14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation. After 12 h incubation, the shape and size of ER was examined using a transmission electron microscope (TEM), and ERS-related genes including immunoglobulin heavy chain binding protein (BiP), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR). Results: After 7, 14 and 21 d of induction, the ALP production, collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT ( P< 0.05). The cells grown on TG, MNT5 and MNT20 surfaces displayed gross distortions of the ER. Compared to PT, BiP, PERK, ATF4 mRNA expression in TG was respectively 1.87±0.10, 2.24±0.35, 1.85±0.14; BiP, ATF4 mRNA expression in MNT5 were respectively 1.27±0.09, 1.25±0.04; BiP, PERK, ATF4 mRNA expression in MNT20 were respectively 1.44±0.09, 2.40±0.60, 1.48±0.05 ( P< 0.05). Conclusions: MNT triggered different degree of ERS, and the activated ERS may promote MNT-induced osteogenic differentiation.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

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Christen, Verena [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Capelle, Martinus [Crucell, P.O. Box 2048, NL-2301 Leiden (Netherlands); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich, Department of Environmental Systems Science, CH-8092 Zürich (Switzerland)

2013-10-15

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL and Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

International Nuclear Information System (INIS)

Christen, Verena; Capelle, Martinus; Fent, Karl

2013-01-01

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL and Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes

Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

NARCIS (Netherlands)

Benham, A.M.; Lith, M. van; Sitia, R.; Braakman, I.|info:eu-repo/dai/nl/073923737

2013-01-01

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide

Endoplasmic reticulum stress is increased in adipose tissue of women with gestational diabetes.

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Stella Liong

Full Text Available Maternal obesity and gestational diabetes mellitus (GDM are two increasingly common and important obstetric complications that are associated with severe long-term health risks to mothers and babies. IL-1β, which is increased in obese and GDM pregnancies, plays an important role in the pathophysiology of these two pregnancy complications. In non-pregnant tissues, endoplasmic (ER stress is increased in diabetes and can induce IL-1β via inflammasome activation. The aim of this study was to determine whether ER stress is increased in omental adipose tissue of women with GDM, and if ER stress can also upregulate inflammasome-dependent secretion of IL-1β. ER stress markers IRE1α, GRP78 and XBP-1s were significantly increased in adipose tissue of obese compared to lean pregnant women. ER stress was also increased in adipose tissue of women with GDM compared to BMI-matched normal glucose tolerant (NGT women. Thapsigargin, an ER stress activator, induced upregulated secretion of mature IL-1α and IL-1β in human omental adipose tissue explants primed with bacterial endotoxin LPS, the viral dsRNA analogue poly(I:C or the pro-inflammatory cytokine TNF-α. Inhibition of capase-1 with Ac-YVAD-CHO resulted in decreased IL-1α and IL-1β secretion, whereas inhibition of pannexin-1 with carbenoxolone suppressed IL-1β secretion only. Treatment with anti-diabetic drugs metformin and glibenclamide also reduced IL-1α and IL-1β secretion in infection and cytokine-primed adipose tissue. In conclusion, this study has demonstrated ER stress to activate the inflammasome in pregnant adipose tissue. Therefore, increased ER stress may contribute towards the pathophysiology of obesity in pregnancy and GDM.

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

OpenAIRE

Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco

2016-01-01

Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and met...

Simvastatin Attenuates the Oxidative Stress, Endothelial Thrombogenicity and the Inducibility of Atrial Fibrillation in a Rat Model of Ischemic Heart Failure

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Kyoung-Im Cho

2014-08-01

Full Text Available Increased atrial oxidative stress has an important role in inducing and maintaining atrial fibrillation (AF, and the activation of the small GTPase Rac1 contributes to the oxidative stress. We investigated the relationship of Rac1, atrial endothelial thromboprotective markers and AF inducibility and if simvastatin has a potential beneficial effect on a myocardial infarction (MI-induced heart failure (HF rat model. Rats were randomized into three groups (shams, MI group and simvastatin treatment group and underwent echocardiography, AF induction studies and left atrial (LA fibrosis analysis. Atrial Rac 1, sodium calcium exchanger (INCX, sarcoplasmic reticulum calcium ATPase (SERCA, endothelial nitric oxide synthase (eNOS and induced nitric oxide synthase (iNOS were measured. AF inducibility, AF duration and LA fibrosis were significantly higher in the MI group (p < 0.001 vs. sham, which were significantly reduced by simvastatin (p < 0.05 vs. MI. The reduced expressions of atrial eNOS, SERCA, thrombomodulin, tissue factor pathway inhibitor and tissue plasminogen activator in the MI group were significantly improved by simvastatin. Furthermore, the increased expression of atrial iNOS, INCX and Rac1 activity were significantly decreased by the simvastatin. Oxidative stress, endothelial dysfunction and thrombogenicity are associated with the promotion of AF in a rat model of ischemic HF. These were associated with increased Rac1 activity, and simvastatin treatment prevents these changes.

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells.

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Norma C Perez-Rosas

Full Text Available The process of Ca2+ release from sarcoplasmic reticulum (SR comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca2+ concentration ([Ca2+]i with a minimal reduction of the free luminal SR [Ca2+] ([Ca2+]FSR. Importantly, active SR Ca2+ ATPases (SERCA pumps are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca2+ binding sites. A new mathematical model was developed that assumes an increasing SR Ca2+ buffering capacity in response to an increase of the luminal SR [Ca2+] that is called Kinetics-on-Demand (KonD model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca2+]FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca2+ binding proteins switch to standard kinetics. The importance of KonD Ca2+ binding properties is twofold: a more efficient Ca2+ release process and that [Ca2+]FSR and Ca2+-bound to SR proteins ([Ca2+]BSR can be regulated separately allowing for Ca2+ release to occur (provided by Ca2+-bound to luminal Ca2+ binding proteins without an initial reduction of the [Ca2+]FSR.

Sensitivity of Rabbit Ventricular Action Potential and Ca2+ Dynamics to Small Variations in Membrane Currents and Ion Diffusion Coefficients

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Yuan Hung Lo

2013-01-01

Full Text Available Little is known about how small variations in ionic currents and Ca2+ and Na+ diffusion coefficients impact action potential and Ca2+ dynamics in rabbit ventricular myocytes. We applied sensitivity analysis to quantify the sensitivity of Shannon et al. model (Biophys. J., 2004 to 5%–10% changes in currents conductance, channels distribution, and ion diffusion in rabbit ventricular cells. We found that action potential duration and Ca2+ peaks are highly sensitive to 10% increase in L-type Ca2+ current; moderately influenced by 10% increase in Na+-Ca2+ exchanger, Na+-K+ pump, rapid delayed and slow transient outward K+ currents, and Cl− background current; insensitive to 10% increases in all other ionic currents and sarcoplasmic reticulum Ca2+ fluxes. Cell electrical activity is strongly affected by 5% shift of L-type Ca2+ channels and Na+-Ca2+ exchanger in between junctional and submembrane spaces while Ca2+-activated Cl−-channel redistribution has the modest effect. Small changes in submembrane and cytosolic diffusion coefficients for Ca2+, but not in Na+ transfer, may alter notably myocyte contraction. Our studies highlight the need for more precise measurements and further extending and testing of the Shannon et al. model. Our results demonstrate usefulness of sensitivity analysis to identify specific knowledge gaps and controversies related to ventricular cell electrophysiology and Ca2+ signaling.

Isoproterenol potentiation of methyl mercury effects in vivo cardiac ATPasees and 3H-dopamine uptake

International Nuclear Information System (INIS)

Ahammad-Sahib, K.I.; Moorthy, K.S.; Cameron, J.A.; Desaiah, D.

1988-01-01

Isoproterenol, a potent B-adrenergic receptor agonist, has been known to produce infarct-like myocardial lesions in rats characterized by swelling of endoplasmic reticulum. The swelling of this system is interpreted as an influx of large amount of extracellular fluid into myocardial cells by disturbances of the electrolyte metabolism. Isoproterenol is employed clinically as a bronchodilator in respiratory disorders and as a stimulant in heart block and cardiogenic shocks. In spite of its clinical use, possible drug-chemical interactions leading to adverse health effects are obvious when individuals on a regular isoproterenol treatment are exposed to an environmental contaminant such as methyl mercury. Consumption of fish and fish products is by far the most significant route of exposure to environmental mercury. In spite of such a possibility, little is know about isoproterenol-methyl mercury interaction. The present study forms the first of this kind to report such interactions and their effects on cardiac membrane bound enzymes such as Na + -K + and Ca 2+ -ATPases. Since Na + -K + ATPase has been implicated in uptake and release processes of catecholamines, the effects were also studied on 3 H-dopamine uptake by sarcoplasmic reticulum. As a prelude to these proposed long-term chronic studies with non-lethal doses in the present report only single and sub-lethal doses were used for a shorter (48h) duration

Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction.

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Yung, Hong Wa; Hemberger, Myriam; Watson, Erica D; Senner, Claire E; Jones, Carolyn P; Kaufman, Randal J; Charnock-Jones, D Stephen; Burton, Graham J

2012-12-01

We recently reported the first evidence of placental endoplasmic reticulum (ER) stress in the pathophysiology of human intrauterine growth restriction. Here, we used a mouse model to investigate potential underlying mechanisms. Eif2s1(tm1RjK) mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1(tm1RjK) placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in the labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97 mg to 82 mg, p growth factor for placental development; indeed, activity in the Pdk1-Akt-mTOR pathways was decreased in Eif2s1(tm1RjK) placentas, indicating loss of Igf2 signalling. Furthermore, we observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2-null mouse. These results demonstrate that ER stress adversely affects placental development, and that modulation of post-translational processing, and hence bioactivity, of secreted growth factors contributes to this effect. Placental dysmorphogenesis potentially affects fetal growth through reduced exchange capacity. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dark matter in the Reticulum II dSph: a radio search

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Regis, Marco; Richter, Laura; Colafrancesco, Sergio

2017-07-01

We present a deep radio search in the Reticulum II dwarf spheroidal (dSph) galaxy performed with the Australia Telescope Compact Array. Observations were conducted at 16 cm wavelength, with an rms sensitivity of 0.01 mJy/beam, and with the goal of searching for synchrotron emission induced by annihilation or decay of weakly interacting massive particles (WIMPs). Data were complemented with observations on large angular scales taken with the KAT-7 telescope. We find no evidence for a diffuse emission from the dSph and we derive competitive bounds on the WIMP properties. In addition, we detect more than 200 new background radio sources. Among them, we show there are two compelling candidates for being the radio counterpart of the possible γ-ray emission reported by other groups using Fermi-LAT data.

Dark matter in the Reticulum II dSph: a radio search

International Nuclear Information System (INIS)

Regis, Marco; Richter, Laura; Colafrancesco, Sergio

2017-01-01

We present a deep radio search in the Reticulum II dwarf spheroidal (dSph) galaxy performed with the Australia Telescope Compact Array. Observations were conducted at 16 cm wavelength, with an rms sensitivity of 0.01 mJy/beam, and with the goal of searching for synchrotron emission induced by annihilation or decay of weakly interacting massive particles (WIMPs). Data were complemented with observations on large angular scales taken with the KAT-7 telescope. We find no evidence for a diffuse emission from the dSph and we derive competitive bounds on the WIMP properties. In addition, we detect more than 200 new background radio sources. Among them, we show there are two compelling candidates for being the radio counterpart of the possible γ-ray emission reported by other groups using Fermi-LAT data.

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

Science.gov (United States)

Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

2017-07-25

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

TRB3 reverses chemotherapy resistance and mediates crosstalk between endoplasmic reticulum stress and AKT signaling pathways in MHCC97H human hepatocellular carcinoma cells.

Science.gov (United States)

Li, Yang; Zhu, Danxi; Hou, Lidan; Hu, Bin; Xu, Min; Meng, Xiangjun

2018-01-01

Tribbles homolog 3 (TRB3), a type of pseudokinase that contains a consensus serine/threonine kinase catalytic core structure, is upregulated in hepatocellular carcinoma. However, the effect of TRB3 expression in hepatocellular carcinoma and the molecular mechanisms underlying TRB3-mediated effects on tumorigenesis in hepatocellular carcinoma have not been fully elucidated. The present study focused on the effect of TRB3 expression in MHCC97H hepatocellular carcinoma cells and investigated the underlying molecular mechanisms in MHCC97H cells. In the present study, it was revealed that TRB3 was significantly overexpressed in the MHCC97H hepatocellular carcinoma cell compared with L-02 normal hepatic cells. Under endoplasmic reticulum (ER) stress induced by thapsigargin and tunicamycin, the levels of TRB3, CCAAT/enhancer binding protein homologous protein (CHOP), protein kinase B (AKT) and phosphorylated (p)AKT expression were upregulated. Furthermore, when the expression of TRB3 was silenced by short hairpin (sh)RNA, the survival of MHCC97H hepatocellular carcinoma cells was increased. Notably, following transduction with lentiviral containing TRB3-shRNA, cell survival also increased after treatment with chemotherapy drug cisplatin. The present study demonstrated that knockdown of CHOP by shRNA was able to reduce TRB3 expression, and the knockdown of TRB3 markedly increased the level of pAKT. TRB3 was overexpressed in MHCC97H hepatocellular carcinoma cells, particularly under endoplasmic reticulum stress. Knockdown of TRB3 was able to increase cell survival. Therefore, TRB3 expression may induce apoptosis and reverse resistance to chemotherapy in MHCC97H hepatic carcinoma cells. The present study suggests that TRB3 is a key molecule that mediates the crosstalk between ER stress and AKT signal pathways. Furthermore, the present study may provide further insight into the cancer biology of hepatocellular carcinoma and the development of anticancer drugs targeting the ER

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

Science.gov (United States)

Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

2016-05-20

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response Constitutes a Pathogenic Strategy of group A Streptococcus

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Emanuel eHanski

2014-08-01

Full Text Available The connection between bacterial pathogens and unfolded protein response (UPR is poorly explored. In this review we highlight the evidence showing that group A streptococcus (GAS induces endoplasmic reticulum (ER stress and UPR through which it captures the amino acid asparagine (ASN from the host. GAS acts extracellularly and during adherence to host cells it delivers the hemolysin toxins; streptolysin O (SLO and streptolysin S (SLS. By poorly understood pathways, these toxins trigger UPR leading to the induction of the transcriptional regulator ATF4 and consequently to the upregulation of asparagine synthetase (ASNS transcription leading to production and release of ASN. GAS senses ASN and alters gene expression profile accordingly, and increases the rate of multiplication. We suggest that induction of UPR by GAS and by other bacterial pathogens represent means through which bacterial pathogens gain nutrients from the host, obviating the need to become internalized or inflict irreversible cell damage.

Friend or foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

Science.gov (United States)

Chen, Shaohua; Zhang, Daohai

2015-01-01

The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as “friend or foe” in epithelial cancer. PMID:25709888

Asymmetrical cross-talk between the endoplasmic reticulum stress and oxidative stress caused by dextrose.

Science.gov (United States)

Mooradian, Arshag D; Onstead-Haas, Luisa; Haas, Michael J

2016-01-01

Oxidative and endoplasmic reticulum (ER) stresses are implicated in premature cardiovascular disease in people with diabetes. The aim of the present study was to characterize the nature of the interplay between the oxidative and ER stresses to facilitate the development of therapeutic agents that can ameliorate these stresses. Human coronary artery endothelial cells were treated with varying concentrations of dextrose in the presence or absence of three antioxidants (alpha tocopherol, ascorbate and ebselen) and two ER stress modifiers (ERSMs) (4-phenylbutyrate and taurodeoxycholic acid). ER stress was measured using the placental alkaline phosphatase assay and superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence. The SO generation was increased with increasing concentrations of dextrose. The ER stress was increased with both low (0 and 2.75 mM) and high (13.75 and 27.5 mM) concentrations of dextrose. The antioxidants inhibited the dextrose induced SO production while in high concentrations they aggravated ER stress. The ERSM reduced ER stress and potentiated the efficacy of the three antioxidants. Tunicamycin-induced ER stress was not associated with increased SO generation. Time course experiments with a high concentration of dextrose or by overexpressing glucose transporter one in endothelial cells revealed that dextrose induced SO generation undergoes adaptive down regulation within 2 h while the ER stress is sustained throughout 72 h of observation. The nature of the cross talk between oxidative stress and ER stress induced by dextrose may explain the failure of antioxidant therapy in reducing diabetes complications. Copyright © 2015 Elsevier Inc. All rights reserved.

ATF4- and CHOP-Dependent Induction of FGF21 through Endoplasmic Reticulum Stress

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Xiao-shan Wan

2014-01-01

Full Text Available Fibroblast growth factor 21 (FGF21 is an important endogenous regulator involved in the regulation of glucose and lipid metabolism. FGF21 expression is strongly induced in animal and human subjects with metabolic diseases, but little is known about the molecular mechanism. Endoplasmic reticulum (ER stress plays an essential role in metabolic homeostasis and is observed in numerous pathological processes, including type 2 diabetes, overweight, nonalcoholic fatty liver disease (NAFLD. In this study, we investigate the correlation between the expression of FGF21 and ER stress. We demonstrated that TG-induced ER stress directly regulated the expression and secretion of FGF21 in a dose- and time-dependent manner. FGF21 is the target gene for activating transcription factor 4 (ATF4 and CCAAT enhancer binding protein homologous protein (CHOP. Suppression of CHOP impaired the transcriptional activation of FGF21 by TG-induced ER stress in CHOP−/− mouse primary hepatocytes (MPH, and overexpression of ATF4 and CHOP resulted in FGF21 promoter activation to initiate the transcriptional programme. In mRNA stability assay, we indicated that ER stress increased the half-life of mRNA of FGF21 significantly. In conclusion, FGF21 expression is regulated by ER stress via ATF- and CHOP-dependent transcriptional mechanism and posttranscriptional mechanism, respectively.

Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

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Zhongxia Wang

2014-01-01

Full Text Available Background. Hepatocellular carcinoma (HCC remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC.

Cadmium-induced teratogenicity: Association with ROS-mediated endoplasmic reticulum stress in placenta

International Nuclear Information System (INIS)

Wang, Zhen; Wang, Hua; Xu, Zhong Mei; Ji, Yan-Li; Chen, Yuan-Hua; Zhang, Zhi-Hui; Zhang, Cheng; Meng, Xiu-Hong; Zhao, Mei; Xu, De-Xiang

2012-01-01

The placenta is essential for sustaining the growth of the fetus. An increased endoplasmic reticulum (ER) stress has been associated with the impaired placental and fetal development. Cadmium (Cd) is a potent teratogen that caused fetal malformation and growth restriction. The present study investigated the effects of maternal Cd exposure on placental and fetal development. The pregnant mice were intraperitoneally injected with CdCl 2 (4.5 mg/kg) on gestational day 9. As expected, maternal Cd exposure during early limb development significantly increased the incidences of forelimb ectrodactyly in fetuses. An obvious impairment in the labyrinth, a highly developed tissue of blood vessels, was observed in placenta of mice treated with CdCl 2 . In addition, maternal Cd exposure markedly repressed cell proliferation and increased apoptosis in placenta. An additional experiment showed that maternal Cd exposure significantly upregulated the expression of GRP78, an ER chaperone. Moreover, maternal Cd exposure induced the phosphorylation of placental eIF2α, a downstream molecule of PERK signaling. In addition, maternal Cd exposure significantly increased the level of placental CHOP, another target of PERK signaling, indicating that the unfolded protein response (UPR) signaling was activated in placenta of mice treated with CdCl 2 . Interestingly, alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, significantly alleviated Cd-induced placental ER stress and UPR. Taken together, these results suggest that reactive oxygen species (ROS)-mediated ER stress might be involved in Cd-induced impairment on placental and fetal development. Antioxidants may be used as pharmacological agents to protect against Cd-induced fetal malformation and growth restriction. -- Highlights: ► Cd induces fetal malformation and growth restriction. ► Cd induced placental ER stress and UPR. ► PBN alleviates Cd-induced ER stress and UPR in placenta. ► ROS-mediated ER stress might

Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

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Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

2011-01-01

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PMID:21857022

Endoplasmic Reticulum Stress Caused by Lipoprotein Accumulation Suppresses Immunity against Bacterial Pathogens and Contributes to Immunosenescence

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Jogender Singh

2017-05-01

Full Text Available The unfolded protein response (UPR is a stress response pathway that is activated upon increased unfolded and/or misfolded proteins in the endoplasmic reticulum (ER, and enhanced ER stress response prolongs life span and improves immunity. However, the mechanism by which ER stress affects immunity remains poorly understood. Using the nematode Caenorhabditis elegans, we show that mutations in the lipoproteins vitellogenins, which are homologs of human apolipoprotein B-100, resulted in upregulation of the UPR. Lipoprotein accumulation in the intestine adversely affects the immune response and the life span of the organism, suggesting that it could be a contributing factor to immunosenescence. We show that lipoprotein accumulation inhibited the expression of several immune genes encoding proteins secreted by the intestinal cells in an IRE-1-independent manner. Our studies provide a mechanistic explanation for adverse effects caused by protein aggregation and ER stress on immunity and highlight the role of an IRE-1-independent pathway in the suppression of the expression of genes encoding secreted proteins.

Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

International Nuclear Information System (INIS)

Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

Energy Technology Data Exchange (ETDEWEB)

Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

2013-10-15

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

Endoplasmic reticulum stress and IRE-1 signaling cause apoptosis in colon cancer cells in response to andrographolide treatment.

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Banerjee, Aditi; Ahmed, Hafiz; Yang, Peixin; Czinn, Steven J; Blanchard, Thomas G

2016-07-05

The plant metabolite andrographolide induces cell cycle arrest and apoptosis in cancer cells. The mechanism(s) by which andrographolide induces apoptosis however, have not been elucidated. The present study was performed to determine the molecular events that promote apoptosis in andrographolide treated cells using T84, HCT116 and COLO 205 colon cancer cell lines. Andrographolide was determined to limit colony formation and Ki67 expression, alter nuclear morphology, increase cytoplasmic histone-associated-DNA-fragments, and increase cleaved caspase-3 levels. Andrographolide also induced significantly higher expression of endoplasmic reticulum (ER) stress proteins GRP-78 and IRE-1 by 48 h but not PERK or ATF6. Apoptosis signaling molecules BAX, spliced XBP-1 and CHOP were also significantly increased. Moreover, chemical inhibition of ER stress or IRE-1 depletion with siRNA in andrographolide treated cells significantly limited expression of IRE-1 and CHOP as determined by immunofluorescence staining, real time PCR, or immunobloting. This was accompanied by a decreased BAX/Bcl-2 ratio. Andrographolide significantly promotes cancer cell death compared to normal cells. These data demonstrate that andrographolide associated ER stress contributes to apoptosis through the activation of a pro-apoptotic GRP-78/IRE-1/XBP-1/CHOP signaling pathway.

Endoplasmic reticulum stress is involved in arsenite-induced oxidative injury in rat brain

International Nuclear Information System (INIS)

Lin, Anya M.Y.; Chao, P.L.; Fang, S.F.; Chi, C.W.; Yang, C.H.

2007-01-01

The mechanism underlying sodium arsenite (arsenite)-induced neurotoxicity was investigated in rat brain. Arsenite was locally infused in the substantia nigra (SN) of anesthetized rat. Seven days after infusion, lipid peroxidation in the infused SN was elevated and dopamine level in the ipsilateral striatum was reduced in a concentration-dependent manner (0.3-5 nmol). Furthermore, local infusion of arsenite (5 nmol) decreased GSH content and increased expression of heat shock protein 70 and heme oxygenase-1 in the infused SN. Aggregation of α-synuclein, a putative pathological protein involved in several CNS neurodegenerative diseases, was elevated in the arsenite-infused SN. From the breakdown pattern of α-spectrin, both necrosis and apoptosis were involved in the arsenite-induced neurotoxicity. Pyknotic nuclei, cellular shrinkage and cytoplasmic disintegration, indicating necrosis, and TUNEL-positive cells and DNA ladder, indicating apoptosis was observed in the arsenite-infused SN. Arsenite-induced apoptosis was mediated via two different organelle pathways, mitochondria and endoplasmic reticulum (ER). For mitochondrial activation, cytosolic cytochrome c and caspase-3 levels were elevated in the arsenite-infused SN. In ER pathway, arsenite increased activating transcription factor-4, X-box binding protein 1, C/EBP homologues protein (CHOP) and cytosolic immunoglobulin binding protein levels. Moreover, arsenite reduced procaspase 12 levels, an ER-specific enzyme in the infused SN. Taken together, our study suggests that arsenite is capable of inducing oxidative injury in CNS. In addition to mitochondria, ER stress was involved in the arsenite-induced apoptosis. Arsenite-induced neurotoxicity clinically implies a pathophysiological role of arsenite in CNS neurodegeneration

Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

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Andrea Janz Moreira

Full Text Available Hepatocellular carcinoma (HCC is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP ribose polymerase (PARP cleavage, and Bcl-associated X protein (Bax/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6, C/EBP-homologous protein (CHOP and immunoglobulin heavy chain-binding protein (BiP, while cyclooxygenase (COX-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

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Mauricio Torres

2010-12-01

Full Text Available Prion-related disorders (PrDs are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES. Altered endoplasmic reticulum (ER homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages

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Fenglei Chen

2015-08-01

Full Text Available Zearalenone (ZEA is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78 and CCAAT/enhancer binding protein homologous protein (CHOP, two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs, significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

DEFF Research Database (Denmark)

Busti, Stefano; Mapelli, Valeria; Tripodi, Farida

2016-01-01

respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections...... reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular...

Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

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Komoike, Yuta, E-mail: komoike@research.twmu.ac.jp; Matsuoka, Masato, E-mail: matsuoka@research.twmu.ac.jp

2016-11-01

Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level of c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. - Highlights: • Exposure of SH-SY5Y cells to AA activates the eIF2α–ATF4 pathway of the UPR. • Exposure of SH-SY5Y cells to AA induces the CHOP expression and apoptosis. • Exposure of zebrafish to AA induces the chop expression and apoptosis in the brain. • AA possibly induces apoptotic neuronal cell death through the ER

Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana

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Thomas eDe Meyer

2014-09-01

Full Text Available A wide variety of recombinant proteins has been produced in the dicot model plant, Arabidopsis thaliana. Many of these proteins are targeted for secretion by means of an N terminal endoplasmic reticulum (ER signal peptide. In addition, they can also be designed for ER retention by adding a C terminal H/KDEL-tag. Despite extensive knowledge of the protein trafficking pathways, the final protein destination, especially of such H/KDEL-tagged recombinant proteins, is unpredictable. In this respect, glycoproteins are ideal study objects. Microscopy experiments reveal their deposition pattern and characterization of their N-glycans aids in elucidating the trafficking. Here, we combine microscopy and N glycosylation data generated in Arabidopsis leaves and seeds, and highlight the lack of a decent understanding of heterologous protein trafficking.

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling.

Science.gov (United States)

Lange, Klaus; Gartzke, Joachim

2006-11-01

A short comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating endoplasmic/sarcoplasmic reticulum-derived vesicles equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive channel-receptors for Ca-release. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of actin filaments. Cellular sites of F-actin-based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling. Copyright 2006 Wiley-Liss, Inc.

Cardiac Dysrhythmias and Neurological Dysregulation: Manifestations of Profound Hypomagnesemia

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Sagger Mawri

2017-01-01

Full Text Available Magnesium is the second most common intracellular cation and serves as an important metabolic cofactor to over 300 enzymatic reactions throughout the human body. Among its various roles, magnesium modulates calcium entry and release from sarcoplasmic reticulum and regulates ATP pumps in myocytes and neurons, thereby regulating cardiac and neuronal excitability. Therefore, deficiency of this essential mineral may result in serious cardiovascular and neurologic derangements. In this case, we present the clinical course of a 76-year-old woman who presented with marked cardiac and neurological signs and symptoms which developed as a result of severe hypomagnesemia. The patient promptly responded to magnesium replacement once the diagnosis was established. We herein discuss the clinical presentation, pathophysiology, diagnosis, and management of severe hypomagnesemia and emphasize the implications of magnesium deficiency in the cardiovascular and central nervous systems. Furthermore, this case highlights the importance of having high vigilance for hypomagnesemia in the appropriate clinical setting.

The destiny of Ca(2+) released by mitochondria.

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Takeuchi, Ayako; Kim, Bongju; Matsuoka, Satoshi

2015-01-01

Mitochondrial Ca(2+) is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca(2+) within mitochondria, Ca(2+) released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca(2+) dynamics have been identified: a mitochondrial Ca(2+) uniporter (MCU), a mitochondrial Na(+)-Ca(2+) exchanger (NCLX), and a candidate for a mitochondrial H(+)-Ca(2+) exchanger (Letm1). In this review, we focus on the mitochondrial Ca(2+) release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca(2+) release system is not only crucial in maintaining mitochondrial Ca(2+) homeostasis but also participates in the Ca(2+) crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.

Gene Therapy in Cardiac Arrhythmias

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Praveen S.V

2006-04-01

Full Text Available Gene therapy has progressed from a dream to a bedside reality in quite a few human diseases. From its first application in adenosine deaminase deficiency, through the years, its application has evolved to vascular angiogenesis and cardiac arrhythmias. Gene based biological pacemakers using viral vectors or mesenchymal cells tested in animal models hold much promise. Induction of pacemaker activity within the left bundle branch can provide stable heart rates. Genetic modification of the AV node mimicking beta blockade can be therapeutic in the management of atrial fibrillation. G protein overexpression to modify the AV node also is experimental. Modification and expression of potassium channel genes altering the delayed rectifier potassium currents may permit better management of congenital long QT syndromes. Arrhythmias in a failing heart are due to abnormal calcium cycling. Potential targets for genetic modulation include the sarcoplasmic reticulum calcium pump, calsequestrin and sodium calcium exchanger.Lastly the ethical concerns need to be addressed.

Exercise-Induced Rhabdomyolysis and Stress-Induced Malignant Hyperthermia Events, Association with Malignant Hyperthermia Susceptibility, and RYR1 Gene Sequence Variations

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Antonella Carsana

2013-01-01

Full Text Available Exertional rhabdomyolysis (ER and stress-induced malignant hyperthermia (MH events are syndromes that primarily afflict military recruits in basic training and athletes. Events similar to those occurring in ER and in stress-induced MH events are triggered after exposure to anesthetic agents in MH-susceptible (MHS patients. MH is an autosomal dominant hypermetabolic condition that occurs in genetically predisposed subjects during general anesthesia, induced by commonly used volatile anesthetics and/or the neuromuscular blocking agent succinylcholine. Triggering agents cause an altered intracellular calcium regulation. Mutations in RYR1 gene have been found in about 70% of MH families. The RYR1 gene encodes the skeletal muscle calcium release channel of the sarcoplasmic reticulum, commonly known as ryanodine receptor type 1 (RYR1. The present work reviews the documented cases of ER or of stress-induced MH events in which RYR1 sequence variations, associated or possibly associated to MHS status, have been identified.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

Science.gov (United States)

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc; Brennen, W Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; Demarzo, Angelo M; Wilding, George; Carducci, Michael A; Dionne, Craig A; Møller, Jesper V; Nissen, Poul; Christensen, S Brøgger; Isaacs, John T

2012-06-27

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

DEFF Research Database (Denmark)

Aschenbach, W G; Suzuki, Y; Breeden, K

2001-01-01

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase...... that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol....... Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction...

Cartap hydrochloride poisoning: A clinical experience.

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Boorugu, Hari K; Chrispal, Anugrah

2012-01-01

Cartap hydrochloride, a nereistoxin analog, is a commonly used low toxicity insecticide. We describe a patient who presented to the emergency department with alleged history of ingestion of Cartap hydrochloride as an act of deliberate self-harm. The patient was managed conservatively. To our knowledge this is the first case report of Cartap hydrochloride suicidal poisoning. Cartap toxicity has been considered to be minimal, but a number of animal models have shown significant neuromuscular toxicity resulting in respiratory failure. It is hypothesized that the primary effect of Cartap hydrochloride is through inhibition of the [(3)H]-ryanodine binding to the Ca(2+) release channel in the sarcoplasmic reticulum in a dose-dependent manner and promotion of extracellular Ca(2+) influx and induction of internal Ca(2+) release. This results in tonic diaphragmatic contraction rather than paralysis. This is the basis of the clinical presentation of acute Cartap poisoning as well as the treatment with chelators namely British Anti Lewisite and sodium dimercaptopropane sulfonate.

The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis*

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Fryer, Lee G. D.; Jones, Bethan; Duncan, Emma J.; Hutchison, Claire E.; Ozkan, Tozen; Williams, Paul A.; Alder, Olivia; Nieuwdorp, Max; Townley, Anna K.; Mensenkamp, Arjen R.; Stephens, David J.; Dallinga-Thie, Geesje M.; Shoulders, Carol C.

2014-01-01

Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions. PMID:24338480

The glutathione mimic ebselen inhibits oxidative stress but not endoplasmic reticulum stress in endothelial cells.

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Ahwach, Salma Makhoul; Thomas, Melanie; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

2015-08-01

Reactive oxygen species are associated with cardiovascular disease, diabetes, and atherosclerosis, yet the use of antioxidants in clinical trials has been ineffective at improving outcomes. In endothelial cells, high-dextrose-induced oxidative stress and endoplasmic reticulum stress promote endothelial dysfunction leading to the recruitment and activation of peripheral blood lymphocytes and the breakdown of barrier function. Ebselen, a glutathione peroxidase 1 (GPX1) mimic, has been shown to improve β-cell function in diabetes and prevent atherosclerosis. To determine if ebselen inhibits both oxidative stress and endoplasmic reticulum (ER) stress in endothelial cells, we examined its effects in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) with and without high-dextrose. Oxidative stress and ER stress were measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence and ER stress alkaline phosphatase assays, respectively. GPX1 over-expression and knockdown were performed by transfecting cells with a GPX1 expression construct or a GPX1-specific siRNA, respectively. Ebselen inhibited dextrose-induced oxidative stress but not ER stress in both HUVEC and HCAEC. Ebselen also had no effect on tunicamycin-induced ER stress in HCAEC. Furthermore, augmentation of GPX1 activity directly by sodium selenite supplementation or transfection of a GPX1 expression plasmid decreased dextrose-induced oxidative stress but not ER stress, while GPX1 knockout enhanced oxidative stress but had no effect on ER stress. These results suggest that ebselen targets only oxidative stress but not ER stress. Copyright © 2015. Published by Elsevier Inc.

Acute fluoride poisoning alters myocardial cytoskeletal and AMPK signaling proteins in rats.

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Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

2017-02-15

Our previous findings revealed that increased oxidative stress, apoptosis and necrosis were implicated in acute fluoride (F - ) induced cardiac dysfunction apart from hypocalcemia and hyperkalemia. Cardiac intermediate filaments (desmin and vimentin) and cytoskeleton linker molecule vinculin plays an imperative role in maintaining the architecture of cardiac cytoskeleton. In addition, AMPK is a stress activated kinase that regulates the energy homeostasis during stressed state. The present study was aimed to examine the role of cytoskeletal proteins and AMPK signaling molecules in acute F - induced cardiotoxicity in rats. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90mg/kgF - for 24h. Acute F - intoxicated rats showed declined cytoskeletal protein expression of desmin, vimentin and vinculin in a dose dependent manner compared to control. A significant increase in phosphorylation of AMPKα (Thr172), AMPKß1 (Ser108) and Acetyl-coA carboxylase (ACC) (Ser79) in the myocardium and associated ATP deprivation were found in acute F - intoxicated rats. Further, ultra-structural studies confirmed myofibril lysis with interruption of Z lines, dilated sarcoplasmic reticulum and damaged mitochondrion were observed in both the groups of F - intoxicated rats. Taken together, these findings reveal that acute F - exposure causes sudden heart failure by altering the expression of cytoskeletal proteins and AMPK signaling molecules. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Functional Effects of Hyperthyroidism on Cardiac Papillary Muscle in Rats.

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Vieira, Fabricio Furtado; Olivoto, Robson Ruiz; Silva, Priscyla Oliveira da; Francisco, Julio Cesar; Fogaça, Rosalvo Tadeu Hochmuller

2016-12-01

Hyperthyroidism is currently recognized to affect the cardiovascular system, leading to a series of molecular and functional changes. However, little is known about the functional influence of hyperthyroidism in the regulation of cytoplasmic calcium and on the sodium/calcium exchanger (NCX) in the cardiac muscle. To evaluate the functional changes in papillary muscles isolated from animals with induced hyperthyroidism. We divided 36 Wistar rats into a group of controls and another of animals with hyperthyroidism induced by intraperitoneal T3 injection. We measured in the animals' papillary muscles the maximum contraction force, speed of contraction (+df/dt) and relaxation (-df/dt), contraction and relaxation time, contraction force at different concentrations of extracellular sodium, post-rest potentiation (PRP), and contraction force induced by caffeine. In hyperthyroid animals, we observed decreased PRP at all rest times (p < 0.05), increased +df/dt and -df/dt (p < 0.001), low positive inotropic response to decreased concentration of extracellular sodium (p < 0.001), reduction of the maximum force in caffeine-induced contraction (p < 0.003), and decreased total contraction time (p < 0.001). The maximal contraction force did not differ significantly between groups (p = 0.973). We hypothesize that the changes observed are likely due to a decrease in calcium content in the sarcoplasmic reticulum, caused by calcium leakage, decreased expression of NCX, and increased expression of a-MHC and SERCA2.

Differences in Ca2+-management between the ventricle of two species of neotropical teleosts: the jeju, Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829, and the acara, Geophagus brasiliensis (Quoy & Gaimard, 1824

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Monica Jones Costa

Full Text Available This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae, and the acara, Geophagus brasiliensis (Cichlidae. While the in vivo heart frequency (fH - bpm of acara (79.6 ± 6.6 was higher than that of the jeju (50.3 ± 2.7, the opposite was observed for the ventricular inotropism (Fc - mN/mm² at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67. A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90% of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20% decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm and physiological (in vivo frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M. In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju, while in acanthopterygians (as acara it seems to be functional mainly in 'athletic' species.

Changes in the distribution of plastids and endoplasmic reticulum during cellular differentiation in root caps of Zea mays

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Moore, R.; McClelen, C. E.

1985-01-01

In calyptrogen cells of Zea mays, proplastids are distributed randomly throughout the cell, and the endoplasmic reticulum (ER) is distributed parallel to the cell walls. The differentiation of calyptrogen cells into columella statocytes is characterized by the following sequential events: (1) formation of ER complexes at the distal and proximal ends of the cell, (2) differentiation of proplastids into amyloplasts, (3) sedimentation of amyloplasts onto the distal ER complex, (4) breakdown of the distal ER complex and sedimentation of amyloplasts to the bottom of the cell, and (5) formation of sheets of ER parallel to the longitudinal cell walls. Columella statocytes located in the centre of the cap each possess 4530 +/- 780 micrometers2 of ER surface area, an increase of 670 per cent over that of calyptrogen cells. The differentiation of peripheral cells correlates positively with (1) the ER becoming arranged in concentric sheets, (2) amyloplasts and ER becoming randomly distributed, and (3) a 280 per cent increase in ER surface area over that of columella statocytes. These results are discussed relative to graviperception and mucilage secretion, which are functions of columella and peripheral cells, respectively.

Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

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Bosquet, Alba; Guaita-Esteruelas, Sandra; Saavedra, Paula; Rodríguez-Calvo, Ricardo; Heras, Mercedes; Girona, Josefa; Masana, Lluís

2016-06-01

Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 μM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. eFABP4, per se, induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

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Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

2016-04-01

Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Arachidonoyl-specific diacylglycerol kinase ε and the endoplasmic reticulum

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Tomoyuki Nakano

2016-11-01

Full Text Available The endoplasmic reticulum (ER comprises an interconnected membrane network, which is made up of lipid bilayer and associated proteins. This organelle plays a central role in the protein synthesis and sorting. In addition, it represents the synthetic machinery of phospholipids, the major constituents of the biological membrane. In this process, phosphatidic acid (PA serves as a precursor of all phospholipids, suggesting that PA synthetic activity is closely associated with the ER function. One enzyme responsible for PA synthesis is diacylglycerol kinase (DGK that phosphorylates diacylglycerol (DG to PA. DGK is composed of a family of enzymes with distinct features assigned to each isozyme in terms of structure, enzymology and subcellular localization. Of DGKs, DGKε uniquely exhibits substrate specificity toward arachidonate-containing DG and is shown to reside in the ER. Arachidonic acid, a precursor of bioactive eicosanoids, is usually acylated at the sn-2 position of phospholipids, being especially enriched in phosphoinositide. In this review, we focus on arachidonoyl-specific DGKε with respect to the historical context, molecular basis of the substrate specificity and ER-targeting, and functional implications in the ER.

The endoplasmic reticulum in plant immunity and cell death.

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Eichmann, Ruth; Schäfer, Patrick

2012-01-01

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants.

The endoplasmic reticulum in plant immunity and cell death

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Patrick eSchäfer

2012-08-01

Full Text Available The endoplasmic reticulum (ER is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programmed cell death (PCD in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signalling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants.

Endoplasmic reticulum (ER Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

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Thomas eSimmen

2014-10-01

Full Text Available Endoplasmic reticulum (ER chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed bulk flow, ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER. However, solid tumors are characterized by the increased production of reactive oxygen species (ROS, combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response (UPR upregulate ER chaperones and oxidoreductases. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the production of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an eat-me signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI, Ero1α and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

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Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

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Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

Effect of Electroacupuncture on the Expression of Glycyl-tRNA Synthetase and Ultrastructure Changes in Atrophied Rat Peroneus Longus Muscle Induced by Sciatic Nerve Injection Injury

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Meng Wang

2016-01-01

Full Text Available Glycyl-tRNA synthetase (GlyRS is one of the key enzymes involved in protein synthesis. Its mutations have been reported to cause Charcot-Marie-Tooth disease which demonstrates muscular atrophy in distal extremities, particularly manifested in peroneus muscles. In this situation, the dysfunctions of mitochondria and sarcoplasmic reticulum (SR affect energy supply and excitation-contraction coupling of muscle fibers, therefore resulting in muscular atrophy. Although the treatment of muscular atrophy is a global urgent problem, it can be improved by electroacupuncture (EA treatment. To investigate the mechanism underlying EA treatment improving muscular atrophy, we focused on the perspective of protein synthesis by establishing a penicillin injection-induced sciatic nerve injury model. In our model, injured rats without treatment showed decreased sciatic functional index (SFI, decreased peroneus longus muscle weight and muscle fiber cross-sectional area, aggregated mitochondria with vacuoles appearing, swollen SR, and downregulated mRNA and protein expression levels of GlyRS and myosin heavy chain IIb (MHC-IIb. The injured rats with EA treatment showed significant recovery. These results indicated that EA stimulation can alleviate peroneus longus muscular atrophy induced by iatrogenic sciatic nerve injury through promoting the recovery of GlyRS and muscle ultrastructure and increasing muscle protein synthesis.

JSA guideline for the management of malignant hyperthermia crisis 2016.

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2017-04-01

Malignant hyperthermia (MH) can be fatal if the crisis is not appropriately treated. It is an inherited disease usually triggered by the administration of volatile inhalational anesthetics and/or succinylcholine, a muscle relaxant. In a patient with suspected MH, the mechanism of calcium release from storage in the sarcoplasmic reticulum in the skeletal muscle is abnormally accelerated. Unexplained hypercarbia representing >55 mmHg of end-tidal carbon dioxide, tachycardia, and muscle rigidity (including masseter muscle rigidity) are early signs of the initiation of MH, because the metabolism is accelerated. The body temperature can rise by >0.5 °C/15 min and may reach ≥40 °C. Respiratory and metabolic acidosis, arrhythmia, cola-colored urine, increased levels of serum potassium, and tented T-waves on electrocardiogram are common and can lead to cardiac arrest. MH should be treated by discontinuation of the triggering agents, administration of intravenous dantrolene (initially 1 mg/kg), and reduction of the body temperature. Early diagnosis and sufficient dantrolene with body temperature reduction are essential to relieve the patient's MH crisis. This guideline in Japanese translation has been posted on the website: http://www.anesth.or.jp/guide/pdf/guideline_akuseikounetsu.pdf .

Pitavastatin-attenuated cardiac dysfunction in mice with dilated cardiomyopathy via regulation of myocardial calcium handling proteins

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Hu Wei

2014-03-01

Full Text Available C57BL/6 mice with dilated cardiomyopathy (DCM were randomly divided to receive placebo or pitavastatin at a dose of 1 or 3 mg kg-1d-1. After 8 weeks treatment, mice with dilated cardiomyopathy developed serious cardiac dysfunction characterized by significantly enhanced left ventricular end-diastolic diameter (LVIDd, decreased left ventricular ejection fraction (LVEF as well as left ventricular short axis fractional shortening (LVFS, accompanied with enlarged cardiomyocytes, and increased plasma levels of N-terminal pro-B type natriuretic peptide (NT-proBNP and plasma angiotensin II (AngII concentration. Moreover, myocardium sarcoplasmic reticulum Ca2+ pump (SERCA-2 activity was decreased. The ratio of phosphorylated phospholamban (PLB to total PLB decreased significantly with the down-regulation of SERCA- -2a and ryanodine receptor (RyR2 expression. Pitavastatin was found to ameliorate the cardiac dysfunction in mice with dilated cardiomyopathy by reversing the changes in the ratios of phosphorylated PLB to total PLB, SERCA-2a and RyR2 via reducing the plasma AngII concentration and the expressions of myocardium angiotensin II type 1 receptor (AT1R and protein kinase C (PKCb2. The possible underlying mechanism might be the regulation of myocardial AT1R-PKCb2-Ca2+ handling proteins.

The Complex Role of Store Operated Calcium Entry Pathways and Related Proteins in the Function of Cardiac, Skeletal and Vascular Smooth Muscle Cells

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Javier Avila-Medina

2018-03-01

Full Text Available Cardiac, skeletal, and smooth muscle cells shared the common feature of contraction in response to different stimuli. Agonist-induced muscle's contraction is triggered by a cytosolic free Ca2+ concentration increase due to a rapid Ca2+ release from intracellular stores and a transmembrane Ca2+ influx, mainly through L-type Ca2+ channels. Compelling evidences have demonstrated that Ca2+ might also enter through other cationic channels such as Store-Operated Ca2+ Channels (SOCCs, involved in several physiological functions and pathological conditions. The opening of SOCCs is regulated by the filling state of the intracellular Ca2+ store, the sarcoplasmic reticulum, which communicates to the plasma membrane channels through the Stromal Interaction Molecule 1/2 (STIM1/2 protein. In muscle cells, SOCCs can be mainly non-selective cation channels formed by Orai1 and other members of the Transient Receptor Potential-Canonical (TRPC channels family, as well as highly selective Ca2+ Release-Activated Ca2+ (CRAC channels, formed exclusively by subunits of Orai proteins likely organized in macromolecular complexes. This review summarizes the current knowledge of the complex role of Store Operated Calcium Entry (SOCE pathways and related proteins in the function of cardiac, skeletal, and vascular smooth muscle cells.

Functional and structural characterization of a eurytolerant calsequestrin from the intertidal teleost Fundulus heteroclitus.

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A Carl Whittington

Full Text Available Calsequestrins (CSQ are high capacity, medium affinity, calcium-binding proteins present in the sarcoplasmic reticulum (SR of cardiac and skeletal muscles. CSQ sequesters Ca²⁺ during muscle relaxation and increases the Ca²⁺-storage capacity of the SR. Mammalian CSQ has been well studied as a model of human disease, but little is known about the environmental adaptation of CSQ isoforms from poikilothermic organisms. The mummichog, Fundulus heteroclitus, is an intertidal fish that experiences significant daily and seasonal environmental fluctuations and is an interesting study system for investigations of adaptation at the protein level. We determined the full-length coding sequence of a CSQ isoform from skeletal muscle of F. heteroclitus (FCSQ and characterized the function and structure of this CSQ. The dissociation constant (K(d of FCSQ is relatively insensitive to changes in temperature and pH, thus indicating that FCSQ is a eurytolerant protein. We identified and characterized a highly conserved salt bridge network in FCSQ that stabilizes the formation of front-to-front dimers, a process critical to CSQ function. The functional profile of FCSQ correlates with the natural history of F. heteroclitus suggesting that the eurytolerant function of FCSQ may be adaptive.

Hypercholesterolemia aggravates myocardial ischemia reperfusion injury via activating endoplasmic reticulum stress-mediated apoptosis.

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Wu, Nan; Zhang, Xiaowen; Jia, Pengyu; Jia, Dalin

2015-12-01

The effect of hypercholesterolemia on myocardial ischemia reperfusion injury (MIRI) is in controversy and the underlying mechanism is still not well understood. In the present study, we firstly detected the effects of hypercholesterolemia on MIRI and the role of endoplasmic reticulum (ER) stress-mediated apoptosis pathway in this process. The infarct size was determined by TTC staining, and apoptosis was measured by the TUNEL method. The marker proteins of ER stress response and ER stress-mediated apoptosis pathway were detected by Western blot. The results showed that high cholesterol diet-induced hypercholesterolemia significantly increased the myocardial infarct size, the release of myocardium enzyme and the ratio of apoptosis, but did not affect the recovery of cardiac function. Moreover, hypercholesterolemia also remarkably up-regulated the expressions of ER stress markers (glucose-regulated protein 78 and calreticulin) and critical molecules in ER stress-mediated apoptosis pathway (CHOP, caspase 12, phospho-JNK). In conclusion, our study demonstrated that hypercholesterolemia enhanced myocardial vulnerability/sensitivity to ischemia reperfusion injury involved in aggravation the ER stress and activation of ER stress-mediated apoptosis pathway and it gave us a new insight into the underlying mechanisms associated with hypercholesterolemia-induced exaggerated MIRI and also provided a novel target for preventing MIRI in the presence of hypercholesterolemia. Copyright © 2015 Elsevier Inc. All rights reserved.

Triggered Firing and Atrial Fibrillation in Transgenic Mice With Selective Atrial Fibrosis Induced by Overexpression of TGF-β1

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Choi, Eue-Keun; Chang, Po-Cheng; Lee, Young-Soo; Lin, Shien-Fong; Zhu, Wuqiang; Maruyama, Mitsunori; Fishbein, Michael C.; Chen, Zhenhui; der Lohe, Michael Rubart-von; Field, Loren J.; Chen, Peng-Sheng

2013-01-01

Background Calcium transient triggered firing (CTTF) is induced by large intracellular calcium (Cai) transient and short action potential duration (APD). We hypothesized that CTTF underlies the mechanisms of early afterdepolarization (EAD) and spontaneous recurrent atrial fibrillation (AF) in transgenic (Tx) mice with overexpression of transforming growth factor β1 (TGF-β1). Methods and Results MHC-TGFcys33ser Tx mice develop atrial fibrosis because of elevated levels of TGF-β1. We studied membrane potential and Cai transients of isolated superfused atria from Tx and wild-type (Wt) littermates. Short APD and persistently elevated Cai transients promoted spontaneous repetitive EADs, triggered activity and spontaneous AF after cessation of burst pacing in Tx but not Wt atria (39% vs. 0%, P=0.008). We were able to map optically 4 episodes of spontaneous AF re-initiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%), which is more severely fibrotic than the left atrium. Ryanodine and thapsigargin inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. Western blotting showed no significant changes of calsequestrin or sarco/endoplasmic reticulum Ca2+-ATPase 2a. Conclusions Spontaneous AF may occur in the Tx atrium because of CTTF, characterized by APD shortening, prolonged Cai transient, EAD and triggered activity. Inhibition of Ca2+ release from the sarcoplasmic reticulum suppressed spontaneous AF. Our results indicate that CTTF is an important arrhythmogenic mechanism in TGF-β1 Tx atria. PMID:22447020

Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress.

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Hosoi, Toru; Yamaguchi, Rie; Noji, Kikuko; Matsuo, Suguru; Baba, Sachiko; Toyoda, Keisuke; Suezawa, Takahiro; Kayano, Takaaki; Tanaka, Shinpei; Ozawa, Koichiro

2014-03-01

Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress-induced leptin resistance, characterized by insensitivity to the actions of the anti-obesity hormone leptin. This result was further supported by flurbiprofen attenuating high-fat diet-induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti-obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity.

Serca2a and Na(+)/Ca(2+) exchanger are involved in left ventricular function following cardiac remodelling of female rats treated with anabolic androgenic steroid.

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Nascimento, Andrews Marques do; Lima, Ewelyne Miranda de; Brasil, Girlandia Alexandre; Caliman, Izabela Facco; Silva, Josiane Fernandes da; Lemos, Virgínia Soares; Andrade, Tadeu Uggere de; Bissoli, Nazaré Souza

2016-06-15

Anabolic-androgenic steroids are misused, including by women, but little is known about the cardiovascular effects of these drugs on women. To evaluated the effects of nandrolone decanoate (ND) and resistive physical exercise on cardiac contractility in young female rats. Female Wistar rats were separated into 4 groups: C (untrained animals); E (animals were submitted to resistance exercise by jumping in water 5 times per week); ND (animals were treated with ND, 20mg/kg/week for 4weeks); and NDE (trained and treated). The haemodynamic parameters (+dP/dtmax, -dP/dtmin and Tau) were assessed in the left ventricle. The heart was collected for histological analyses and collagen deposition. The gastrocnemius muscle was weighed, and hypertrophy was assessed by the ratio of their weights to gastrocnemius/tibia length. The expression of calcium handling proteins was measured by western blot analysis. ND treatment and physical exercise increased cardiac contractility and relaxation. In addition, ND promoted increases in phospholamban phosphorylated (p-PLB) and isoforms of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, while resistance exercise increased the phosphorylation of PLB and expression of Na(+)/Ca(2+) exchangers (NCX). Cardiac hypertrophy and collagen deposition were observed after ND treatment. Regulatory components of cytosolic calcium, such as SERCA2a and p-PLB, play important roles in modulating the contractility and relaxation effects of ND in females. Copyright © 2016 Elsevier Inc. All rights reserved.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

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Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

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Xue Yu

Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

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Winter, James; Bishop, Martin J; Wilder, Catherine D E; O'Shea, Christopher; Pavlovic, Davor; Shattock, Michael J

2018-01-01

into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart. Conclusions : SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased I Ks .

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding.

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Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J

2010-10-01

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.

Endoplasmic reticulum and lysosomal Ca²⁺ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts.

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Kilpatrick, Bethan S; Magalhaes, Joana; Beavan, Michelle S; McNeill, Alisdair; Gegg, Matthew E; Cleeter, Michael W J; Bloor-Young, Duncan; Churchill, Grant C; Duchen, Michael R; Schapira, Anthony H; Patel, Sandip

2016-01-01

Mutations in β-glucocerebrosidase (encoded by GBA1) cause Gaucher disease (GD), a lysosomal storage disorder, and increase the risk of developing Parkinson disease (PD). The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca(2+) release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in β-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca(2+) release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca(2+) signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of β-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca(2+) signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca(2+) store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca(2+) stores by pathogenic GBA1 mutations may therefore feature in PD. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Endoplasmic reticulum (ER) stress and cAMP/PKA pathway mediated Zn-induced hepatic lipolysis.

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Song, Yu-Feng; Hogstrand, Christer; Wei, Chuan-Chuan; Wu, Kun; Pan, Ya-Xiong; Luo, Zhi

2017-09-01

The present study was performed to determine the effect of Zn exposure influencing endoplasmic reticulum (ER) stress, explore the underlying molecular mechanism of Zn-induced hepatic lipolysis in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco. We found that waterborne Zn exposure evoked ER stress and unfolded protein response (UPR), and activated cAMP/PKA pathway, and up-regulated hepatic lipolysis. The increase in ER stress and lipolysis were associated with activation of cAMP/PKA signaling pathway. Zn also induced an increase in intracellular Ca 2+ level, which could be partially prevented by dantrolene (RyR receptor inhibitor) and 2-APB (IP3 receptor inhibitor), demonstrating that the disturbed Ca 2+ homeostasis in ER contributed to ER stress and dysregulation of lipolysis. Inhibition of ER stress by PBA attenuated UPR, inhibited the activation of cAMP/PKA pathway and resulted in down-regulation of lipolysis. Inhibition of protein kinase RNA-activated-like ER kinase (PERK) by GSK2656157 and inositol-requiring enzyme (IRE) by STF-083010 differentially influenced Zn-induced changes of lipid metabolism, indicating that PERK and IRE pathways played different regulatory roles in Zn-induced lipolysis. Inhibition of PKA by H89 blocked the Zn-induced activation of cAMP/PKA pathway with a concomitant inhibition of ER stress-mediated lipolysis. Taken together, our findings highlight the importance of the ER stress-cAMP/PKA axis in Zn-induced lipolysis, which provides new insights into Zn toxicology in fish and probably in other vertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Streptozotocin alters glucose transport, connexin expression and endoplasmic reticulum functions in neurons and astrocytes.

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Biswas, Joyshree; Gupta, Sonam; Verma, Dinesh Kumar; Singh, Sarika

2017-07-25

The study was undertaken to explore the cell-specific streptozotocin (STZ)-induced mechanistic alterations. STZ-induced rodent model is a well-established experimental model of Alzheimer's disease (AD) and in our previous studies we have established it as an in vitro screening model of AD by employing N2A neuronal cells. Therefore, STZ was selected in the present study to understand the STZ-induced cell-specific alterations by utilizing neuronal N2A and astrocytes C6 cells. Both neuronal and astrocyte cells were treated with STZ at 10, 50, 100 and 1000μM concentrations for 48h. STZ exposure caused significant decline in cellular viability and augmented cytotoxicity of cells involving astrocytes activation. STZ treatment also disrupted the energy metabolism by altered glucose uptake and its transport in both cells as reflected with decreased expression of glucose transporters (GLUT) 1/3. The consequent decrease in ATP level and decreased mitochondrial membrane potential was also observed in both the cells. STZ caused increased intracellular calcium which could cause the initiation of endoplasmic reticulum (ER) stress. Significant upregulation of ER stress-related markers were observed in both cells after STZ treatment. The cellular communication of astrocytes and neurons was altered as reflected by increased expression of connexin 43 along with DNA fragmentation. STZ-induced apoptotic death was evaluated by elevated expression of caspase-3 and PI/Hoechst staining of cells. In conclusion, study showed that STZ exert alike biochemical alterations, ER stress and cellular apoptosis in both neuronal and astrocyte cells. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes.

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Ji, Yan-Li; Wang, Hua; Zhang, Cheng; Zhang, Ying; Zhao, Mei; Chen, Yuan-Hua; Xu, De-Xiang

2013-03-01

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0 mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells

International Nuclear Information System (INIS)

Hagen, Christoph; Werner, Stephan; Carregal-Romero, Susana; Malhas, Ashraf N.; Klupp, Barbara G.; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; Mettenleiter, Thomas C.

2014-01-01

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. - Highlights: • Nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated cells. • New polyelectrolyte-Qdot ® 605 coated gold beads were employed as fiducials. • Saquinavir can induce a strong apoptotic phenotype in the nucleus. • CryoXT is an auspicious imaging technique in apoptosis research

Antiarrhythmic Effects of Dantrolene in Patients with Catecholaminergic Polymorphic Ventricular Tachycardia and Replication of the Responses Using iPSC Models.

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Kirsi Penttinen

Full Text Available Catecholaminergic polymorphic ventricular tachycardia (CPVT is a highly malignant inherited arrhythmogenic disorder. Type 1 CPVT (CPVT1 is caused by cardiac ryanodine receptor (RyR2 gene mutations resulting in abnormal calcium release from sarcoplasmic reticulum. Dantrolene, an inhibitor of sarcoplasmic Ca(2+ release, has been shown to rescue this abnormal Ca(2+ release in vitro. We assessed the antiarrhythmic efficacy of dantrolene in six patients carrying various RyR2 mutations causing CPVT. The patients underwent exercise stress test before and after dantrolene infusion. Dantrolene reduced the number of premature ventricular complexes (PVCs on average by 74% (range 33-97 in four patients with N-terminal or central mutations in the cytosolic region of the RyR2 protein, while dantrolene had no effect in two patients with mutations in or near the transmembrane domain. Induced pluripotent stem cells (iPSCs were generated from all the patients and differentiated into spontaneously beating cardiomyocytes (CMs. The antiarrhythmic effect of dantrolene was studied in CMs after adrenaline stimulation by Ca(2+ imaging. In iPSC derived CMs with RyR2 mutations in the N-terminal or central region, dantrolene suppressed the Ca(2+ cycling abnormalities in 80% (range 65-97 of cells while with mutations in or near the transmembrane domain only in 23 or 32% of cells. In conclusion, we demonstrate that dantrolene given intravenously shows antiarrhythmic effects in a portion of CPVT1 patients and that iPSC derived CM models replicate these individual drug responses. These findings illustrate the potential of iPSC models to individualize drug therapy of inherited diseases.Trial Registration: EudraCT Clinical Trial Registry 2012-005292-14.

Alternagin-C (ALT-C), a disintegrin-like protein from Rhinocerophis alternatus snake venom promotes positive inotropism and chronotropism in fish heart.

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Monteiro, D A; Kalinin, A L; Selistre-de-Araujo, H S; Vasconcelos, E S; Rantin, F T

2016-02-01

Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg(-1), via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca(2+)-cycling proteins such as SERCA (sarcoplasmic reticulum Ca(2+)-ATPase), PLB (phospholamban), and NCX (Na(+)/Ca(2+) exchanger); 4) abolishment of the typical negative force-frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca(2+) handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluoride induces endoplasmic reticulum stress and inhibits protein synthesis and secretion.

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Sharma, Ramaswamy; Tsuchiya, Masahiro; Bartlett, John D

2008-09-01

Exposure to excessive amounts of fluoride (F(-)) causes dental fluorosis in susceptible individuals; however, the mechanism of F(-)-induced toxicity is unclear. Previously, we have shown that high-dose F(-) activates the unfolded protein response (UPR) in ameloblasts that are responsible for dental enamel formation. The UPR is a signaling pathway responsible for either alleviating endoplasmic reticulum (ER) stress or for inducing apoptosis of the stressed cells. In this study we determined if low-dose F(-) causes ER stress and activates the UPR, and we also determined whether F(-) interferes with the secretion of proteins from the ER. We stably transfected the ameloblast-derived LS8 cell line with secreted alkaline phosphatase (SEAP) and determined activity and localization of SEAP and F(-)-mediated induction of UPR proteins. Also, incisors from mice given drinking water containing various concentrations of F(-) were examined for eucaryotic initiation factor-2, subunit alpha (eIF2alpha) phosphorylation. We found that F(-) decreases the extracellular secretion of SEAP in a linear, dose-dependent manner. We also found a corresponding increase in the intracellular accumulation of SEAP after exposure to F(-). These changes are associated with the induction of UPR proteins such as the molecular chaperone BiP and phosphorylation of the UPR sensor PKR-like ER kinase, and its substrate, eIF2alpha. Importantly, F(-)-induced phosphorylation of eIF2alphawas confirmed in vivo. These data suggest that F(-) initiates an ER stress response in ameloblasts that interferes with protein synthesis and secretion. Consequently, ameloblast function during enamel development may be impaired, and this may culminate in dental fluorosis.

Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

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Guo, Haiqing; Ren, Feng; Zhang, Li; Zhang, Xiangying; Yang, Rongrong; Xie, Bangxiang; Li, Zhuo; Hu, Zhongjie; Duan, Zhongping; Zhang, Jing

2016-03-01

Kaempferol is a flavonoid compound that has gained importance due to its antitumor properties; however, the underlying mechanisms remain to be fully understood. The present study aimed to investigate the molecular mechanisms of the antitumor function of kaempferol in HepG2 hepatocellular carcinoma cells. Kaempferol was determined to reduce cell viability, increase lactate dehydrogenase activity and induce apoptosis in a concentration‑ and time‑dependent manner in HepG2 cells. Additionally, kaempferol‑induced apoptosis possibly acts via the endoplasmic reticulum (ER) stress pathway, due to the significant increase in the protein expression levels of glucose‑regulated protein 78, glucose‑regulated protein 94, protein kinase R‑like ER kinase, inositol‑requiring enzyme 1α, partial activating transcription factor 6 cleavage, caspase‑4, C/EBP homologous protein (CHOP) and cleaved caspase‑3. The pro‑apoptotic activity of kaempferol was determined to be due to induction of the ER stress‑CHOP pathway, as: i) ER stress was blocked by 4‑phenyl butyric acid (4‑PBA) pretreatment and knockdown of CHOP with small interfering RNA, which resulted in alleviation of kaempferol‑induced HepG2 cell apoptosis; and ii) transfection with plasmid overexpressing CHOP reversed the protective effect of 4‑PBA in kaempferol‑induced HepG2 cells and increased the apoptotic rate. Thus, kaempferol promoted HepG2 cell apoptosis via induction of the ER stress‑CHOP signaling pathway. These observations indicate that kaempferol may be used as a potential chemopreventive treatment strategy for patients with hepatocellular carcinoma.

Ursodeoxycholic Acid Attenuates Endoplasmic Reticulum Stress-Related Retinal Pericyte Loss in Streptozotocin-Induced Diabetic Mice

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Yoo-Ri Chung

2017-01-01

Full Text Available Loss of pericytes, an early hallmark of diabetic retinopathy (DR, results in breakdown of the blood-retinal barrier. Endoplasmic reticulum (ER stress may be involved in this process. The purpose of this study was to examine the effects of ursodeoxycholic acid (UDCA, a known ameliorator of ER stress, on pericyte loss in DR of streptozotocin- (STZ- induced diabetic mice. To assess the extent of DR, the integrity of retinal vessels and density of retinal capillaries in STZ-induced diabetic mice were evaluated. Additionally, induction of ER stress and the unfolded protein response (UPR were assessed in diabetic mice and human retinal pericytes exposed to advanced glycation end products (AGE or modified low-density lipoprotein (mLDL. Fluorescein dye leakage during angiography and retinal capillary density were improved in UDCA-treated diabetic mice, compared to the nontreated diabetic group. Among the UPR markers, those involved in the protein kinase-like ER kinase (PERK pathway were increased, while UDCA attenuated UPR in STZ-induced diabetic mice as well as AGE- or mLDL-exposed retinal pericytes in culture. Consequently, vascular integrity was improved and pericyte loss reduced in the retina of STZ-induced diabetic mice. Our findings suggest that UDCA might be effective in protecting against DR.

Deletion of an Endoplasmic Reticulum Stress Response Element in a ZmPP2C-A Gene Facilitates Drought Tolerance of Maize Seedlings.

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Xiang, Yanli; Sun, Xiaopeng; Gao, Shan; Qin, Feng; Dai, Mingqiu

2017-03-06

Drought is a major abiotic stress that causes the yearly yield loss of maize, a crop cultured worldwide. Breeding drought-tolerant maize cultivars is a priority requirement of world agriculture. Clade A PP2C phosphatases (PP2C-A), which are conserved in most plant species, play important roles in abscisic acid (ABA) signaling and plant drought response. However, natural variations of PP2C-A genes that are directly associated with drought tolerance remain to be elucidated. Here, we conducted a candidate gene association analysis of the ZmPP2C-A gene family in a maize panel consisting of 368 varieties collected worldwide, and identified a drought responsive gene ZmPP2C-A10 that is tightly associated with drought tolerance. We found that the degree of drought tolerance of maize cultivars negatively correlates with the expression levels of ZmPP2C-A10. ZmPP2C-A10, like its Arabidopsis orthologs, interacts with ZmPYL ABA receptors and ZmSnRK2 kinases, suggesting that ZmPP2C-A10 is involved in mediating ABA signaling in maize. Transgenic studies in maize and Arabidopsis confirmed that ZmPP2C-A10 functions as a negative regulator of drought tolerance. Further, a causal natural variation, deletion allele-338, which bears a deletion of ERSE (endoplasmic reticulum stress response element) in the 5'-UTR region of ZmPP2C-A10, was detected. This deletion causes the loss of endoplasmic reticulum (ER) stress-induced expression of ZmPP2C-A10, leading to increased plant drought tolerance. Our study provides direct evidence linking ER stress signaling with drought tolerance and genetic resources that can be used directly in breeding drought-tolerant maize cultivars. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

OpenAIRE

Zhu, Xueping; Xiao, Zhihui; Xu, Yumin; Zhao, Xingli; Cheng, Ping; Cui, Ningxun; Cui, Mingling; Li, Jie; Zhu, Xiaoli

2016-01-01

Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resu...

Induction of Liver Steatosis and Lipid Droplet Formation in ATF6α-Knockout Mice Burdened with Pharmacological Endoplasmic Reticulum Stress

OpenAIRE

Harada, Akihiro; Okada, Tetsuya; Sato, Takashi; Mori, Kazutoshi; Yamamoto, Keisuke; Takahara, Kazuna; Oyadomari, Seiichi

2010-01-01

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6? plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6? are sensitive to ER s...

3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

International Nuclear Information System (INIS)

Reynaud, S.; Duchiron, C.; Deschaux, P.

2003-01-01

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

Lipocalin 2, a new GADD153 target gene, as an apoptosis inducer of endoplasmic reticulum stress in lung cancer cells

International Nuclear Information System (INIS)

Hsin, I-Lun; Hsiao, Yueh-Chieh; Wu, Ming-Fang; Jan, Ming-Shiou; Tang, Sheau-Chung; Lin, Yu-Wen; Hsu, Chung-Ping; Ko, Jiunn-Liang

2012-01-01

Endoplasmic reticulum (ER) stress is activated under severe cellular conditions. GADD153, a member of the C/EBP family, is an unfolded protein response (UPR) responsive transcription factor. Increased levels of lipocalin 2, an acute phase protein, have been found in several epithelial cancers. The aim of this study is to investigate the function of lipocalin 2 in lung cancer cells under ER stress. Treatment with thapsigargin, an ER stress activator, led to increases in cytotoxicity, ER stress, apoptosis, and lipocalin 2 expression in A549 cells. GADD153 silencing decreased lipocalin 2 expression in A549 cells. On chromatin immunoprecipitation assay, ER stress increased GADD153 DNA binding to lipocalin 2 promoter. Furthermore, silencing of lipocalin 2 mitigated ER stress-mediated apoptosis in A549 cells. Our findings demonstrated that lipocalin 2 is a new GADD153 target gene that mediates ER stress-induced apoptosis. Highlights: ► We demonstrate that Lipocalin 2 is a new GADD153 target gene. ► Lipocalin 2 mediates ER stress-induced apoptosis. ► ER stress-induced lipocalin 2 expression is calcium-independent in A549 cells. ► Lipocalin 2 dose not play a major role in ER stress-induced autophagy.

A mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress

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Sian E. Piret

2017-06-01

Full Text Available Renal fibrosis is a common feature of renal failure resulting from multiple etiologies, including diabetic nephropathy, hypertension and inherited renal disorders. However, the mechanisms of renal fibrosis are incompletely understood and we therefore explored these by establishing a mouse model for a renal tubular disorder, referred to as autosomal dominant tubulointerstitial kidney disease (ADTKD due to missense uromodulin (UMOD mutations (ADTKD-UMOD. ADTKD-UMOD, which is associated with retention of mutant uromodulin in the endoplasmic reticulum (ER of renal thick ascending limb cells, is characterized by hyperuricemia, interstitial fibrosis, inflammation and renal failure, and we used targeted homologous recombination to generate a knock-in mouse model with an ADTKD-causing missense cysteine to arginine uromodulin mutation (C125R. Heterozygous and homozygous mutant mice developed reduced uric acid excretion, renal fibrosis, immune cell infiltration and progressive renal failure, with decreased maturation and excretion of uromodulin, due to its retention in the ER. The ER stress marker 78 kDa glucose-regulated protein (GRP78 was elevated in cells expressing mutant uromodulin in heterozygous and homozygous mutant mice, and this was accompanied, both in vivo and ex vivo, by upregulation of two unfolded protein response pathways in primary thick ascending limb cells from homozygous mutant mice. However, this did not lead to an increase in apoptosis in vivo. Thus, we have developed a novel mouse model for renal fibrosis, which will be a valuable resource to decipher the mechanisms linking uromodulin mutations with ER stress and renal fibrosis.

Melatonin Modulates Neuronal Cell Death Induced by Endoplasmic Reticulum Stress under Insulin Resistance Condition.

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Song, Juhyun; Kim, Oh Yoen

2017-06-10

Insulin resistance (IR) is an important stress factor in the central nervous system, thereby aggravating neuropathogenesis and triggering cognitive decline. Melatonin, which is an antioxidant phytochemical and synthesized by the pineal gland, has multiple functions in cellular responses such as apoptosis and survival against stress. This study investigated whether melatonin modulates the signaling of neuronal cell death induced by endoplasmic reticulum (ER) stress under IR condition using SH-SY5Y neuroblastoma cells. Apoptosis cell death signaling markers (cleaved Poly [ADP-ribose] polymerase 1 (PARP), p53, and Bax) and ER stress markers (phosphorylated eIF2α (p-eIF2α), ATF4, CHOP, p-IRE1 , and spliced XBP1 (sXBP1)) were measured using reverse transcription-PCR, quantitative PCR, and western blottings. Immunofluorescence staining was also performed for p-ASK1 and p-IRE1 . The mRNA or protein expressions of cell death signaling markers and ER stress markers were increased under IR condition, but significantly attenuated by melatonin treatment. Insulin-induced activation of ASK1 ( p-ASK1 ) was also dose dependently attenuated by melatonin treatment. The regulatory effect of melatonin on neuronal cells under IR condition was associated with ASK1 signaling. In conclusion, the result suggested that melatonin may alleviate ER stress under IR condition, thereby regulating neuronal cell death signaling.

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

Science.gov (United States)

Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

2003-07-30

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

Thiamine Deficiency and Neurodegeneration: the Interplay Among Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy.

Science.gov (United States)

Liu, Dexiang; Ke, Zunji; Luo, Jia

2017-09-01

Thiamine (vitamin B1) is an essential nutrient and indispensable for normal growth and development of the organism due to its multilateral participation in key biochemical and physiological processes. Humans must obtain thiamine from their diet since it is synthesized only in bacteria, fungi, and plants. Thiamine deficiency (TD) can result from inadequate intake, increased requirement, excessive deletion, and chronic alcohol consumption. TD affects multiple organ systems, including the cardiovascular, muscular, gastrointestinal, and central and peripheral nervous systems. In the brain, TD causes a cascade of events including mild impairment of oxidative metabolism, neuroinflammation, and neurodegeneration, which are commonly observed in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Thiamine metabolites may serve as promising biomarkers for neurodegenerative diseases, and thiamine supplementations exhibit therapeutic potential for patients of some neurodegenerative diseases. Experimental TD has been used to model aging-related neurodegenerative diseases. However, to date, the cellular and molecular mechanisms underlying TD-induced neurodegeneration are not clear. Recent research evidence indicates that TD causes oxidative stress, endoplasmic reticulum (ER) stress, and autophagy in the brain, which are known to contribute to the pathogenesis of various neurodegenerative diseases. In this review, we discuss the role of oxidative stress, ER stress, and autophagy in TD-mediated neurodegeneration. We propose that it is the interplay of oxidative stress, ER stress, and autophagy that contributes to TD-mediated neurodegeneration.

[Effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma].

Science.gov (United States)

Tian, Jing; Hu, Xiaoming; Qu, Quanxin

2014-05-01

The study intended to investigate the effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma. RT-PCR and Western blot were used to test the expression of mTOR and Beclin1 mRNA and protein in ovarian cancer SKOV3 cells after saquinavir induction. MTT assay was used to analyze the influence of saquinavir on cisplatin sensitivity in SKOV3 cells. The IC50 of SKOV3 cells was (5.490 ± 1.148) µg/ml. After induced by Saquinavair 10 µmol/L and 20 µmol/L, the IC50 of SKOV3 cells was increased to (11.199 ± 0.984) µg/ml and (14.906 ± 2.015) µg/ml, respectively. It suggested that the sensitivity of ovarian cancer cells to cisplatin was decreased significantly (P = 0.001). The expression of mTOR and Beclin1 mRNA and protein was significantly different among the five groups: the (Saquinavair+DDP) group of, Saquinavair group, LY294002 group, DDP group and control group (P cisplatin sensitivity in the SKOV3 cells after Saquinavir induced ER stress (P cisplatin in SKOV3 cells. The mechanism of the decrease of sensitivity to cisplatin in SKOV3 cells may be that ERS regulates cell autophagy through the mTOR and Beclin1 pathways. ERS of tumor cells and autophagy may become a new target to improve the therapeutic effect of chemotherapy and to reverse the drug resistance in tumor treatment.

Mutant tamm-horsfall glycoprotein accumulation in endoplasmic reticulum induces apoptosis reversed by colchicine and sodium 4-phenylbutyrate.

Science.gov (United States)

Choi, Sung Won; Ryu, Ok Hee; Choi, Sun Jin; Song, In Sun; Bleyer, Anthony J; Hart, Thomas C

2005-10-01

As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.