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Sample records for atp induced vasodilatation

  1. ATP induced vasodilatation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins and adenosine

    DEFF Research Database (Denmark)

    Mortensen, Stefan P; Gonzalez-Alonso, Jose; Bune, Laurids

    2009-01-01

    Plasma adenosine-5'-triphosphate (ATP) is thought to contribute to the local regulation of skeletal muscle blood flow. Intravascular ATP infusion can induce profound limb muscle vasodilatation, but the purinergic receptors and downstream signals involved in this response remain unclear. This study...... investigated: 1) the role of nitric oxide (NO), prostaglandins and adenosine as mediators of ATP induced limb vasodilation and 2) the expression and distribution of purinergic P2 receptors in human skeletal muscle. Systemic and leg hemodynamics were measured before and during 5-7 min of femoral intra.......05) and was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O2 extraction. Infusion of theophylline did not alter the ATP induced leg hyperemia or systemic variables. Real time PCR analysis of the mRNA content from the vastus lateralus muscle of 8...

  2. Chronic hypoxia increases arterial blood pressure and reduces adenosine and ATP induced vasodilatation in skeletal muscle in healthy humans

    DEFF Research Database (Denmark)

    Calbet, J A L; Boushel, Robert Christopher; Robach, P

    2014-01-01

    AIMS: To determine the role played by adenosine, ATP and chemoreflex activation on the regulation of vascular conductance in chronic hypoxia. METHODS: The vascular conductance response to low and high doses of adenosine and ATP was assessed in ten healthy men. Vasodilators were infused into the f...

  3. Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine.

    Science.gov (United States)

    Murrant, Coral L; Dodd, Jason D; Foster, Andrew J; Inch, Kristin A; Muckle, Fiona R; Ruiz, Della A; Simpson, Jeremy A; Scholl, Jordan H P

    2014-03-15

    Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 μm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10(-7)-10(-5) M) extraluminally, (to mimic muscle contraction) in the absence and presence of L-NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and L-NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). L-NAME, INDO and L-NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in

  4. Role of KATP channels in cephalic vasodilatation induced by calcitonin gene-related peptide, nitric oxide, and transcranial electrical stimulation in the rat

    DEFF Research Database (Denmark)

    Gozalov, Aydin; Jansen-Olesen, Inger; Klærke, Dan Arne

    2008-01-01

    arteries causes a throbbing, migraine-like pain. Both CGRP and NO are potent vasodilators that can induce migraine. Their antagonists are effective in the treatment of migraine attacks. K(ATP) channel openers cause headache in the majority of healthy subjects suggesting a role for K(ATP) channels......OBJECTIVE: The objective of this study was to explore the role of K(ATP) channels in vasodilatation induced by calcitonin gene-related peptide (CGRP), nitric oxide (NO), and transcranial electrical stimulation (TES) in intracranial arteries of rat. BACKGROUND: Dilatation of cerebral and dural...... in migraine pathogenesis. We hypothesized that vasodilatation induced by CGRP and the NO donor glyceryltrinitrate (GTN) is mediated via K(ATP) channels. METHODS: We examined the effects of the K(ATP) channel inhibitor glibenclamide on dural and pial vasodilatation induced by CGRP, NO, and endogenously...

  5. Levobupivacaine induces vasodilatation, but not vasoconstriction, in rat mesenteric artery

    Directory of Open Access Journals (Sweden)

    Liciane dos Santos MENEZES

    Full Text Available Abstract Introduction Levobupivacaine (LEVO can replace analgesia because it exhibits low toxicity and causes minor vasoconstriction, enabling its use in patients in whom vasoconstrictors are contraindicated. Objective We aimed to evaluate the effects of LEVO in isolated rat superior mesenteric artery by using the vascular reactivity technique and compare its effect to that of lidocaine. Material and method Arterial rings were obtained from the mesenteric artery of male Wistar rats and kept in organ baths. For recording isometric contractions, each ring was suspended by cotton threads from a force transducer, which was connected to a data acquisition system. Result Both lidocaine and LEVO did not show a vasoconstrictor effect on the basal tone of the arterial rings with functional endothelium. However, when the rings were pre-contracted with phenylephrine, both drugs were able to induce concentration-dependent vasodilatation. The vasodilator effect induced by LEVO did not change after removal of the endothelium, or with the addition of tetraethylammonium (1 mM, a non-selective K+ channel blocker. In the rings without functional endothelium, which were pre-contracted with depolarizing Tyrode’s solution (KCl 80 mM, LEVO-induced vasodilatation was not significantly different from that observed in the rings pre-contracted with phenylephrine. Moreover, it did not show a significant additional vasodilator effect compared to the maximal vasodilator effect of nifedipine. Conclusion This study demonstrated that LEVO produces a vasodilator effect in the rat superior mesenteric artery in an endothelium-independent manner. This effect seems to be mediated via Ca2+ channel blockade in the vascular smooth muscle cells.

  6. HYPOTHESIS ON CELLULAR ATP DEPLETION AND ADENOSINE RELEASE AS CAUSES OF HEART-FAILURE AND VASODILATATION IN CARDIOVASCULAR BERIBERI

    NARCIS (Netherlands)

    Bakker, sjl; LEUNISSEN, KML

    Cardiovascular beriberi is a syndrome caused by thiamine deficiency and characterized by systemic vasodilatation, heart failure and lactic acidosis. The occurrence of heart failure and vasodilatation is yet unexplained: neither theoretical nor experimental data are known. In this article, it is

  7. Cold-induced vasodilatation in cold-intolerant rats after nerve injury

    NARCIS (Netherlands)

    Smits, E.S.; Duraku, L.S.; Niehof, S.P.; Daanen, H.A.M.; Hovius, S.E.R.; Selles, R.W.; Walbeehm, E.T.

    2013-01-01

    Purpose Cold-induced vasodilatation (CIVD) is a cyclic regulation of blood flow during prolonged cooling of protruding body parts. It is generally considered to be a protective mechanism against local cold injuries and cold intolerance after peripheral nerve injury. The aim of this study was to

  8. Cold-induced vasodilatation in cold-intolerant rats after nerve injury

    NARCIS (Netherlands)

    Smits, E.S.; Duraku, L.S.; Niehof, S.P.; Daanen, H.A.M.; Hovius, S.E.R.; Selles, R.W.; Walbeehm, E.T.

    2013-01-01

    Summary Purpose: Cold-induced vasodilatation (CIVD) is a cyclic regulation of blood flow during prolonged cooling of protruding body parts. It is generally considered to be a protective mechanism against local cold injuries and cold intolerance after peripheral nerve injury. The aim of this study

  9. Fluoxetine induces vasodilatation of cerebral arterioles by co-modulating NO/muscarinic signalling

    Science.gov (United States)

    Ofek, Keren; Schoknecht, Karl; Melamed-Book, Naomi; Heinemann, Uwe; Friedman, Alon; Soreq, Hermona

    2012-01-01

    Ischaemic stroke patients treated with Selective Serotonin Reuptake Inhibitors (SSRI) show improved motor, cognitive and executive functions, but the underlying mechanism(s) are incompletely understood. Here, we report that cerebral arterioles in the rat brain superfused with therapeutically effective doses of the SSRI fluoxetine showed consistent, dose-dependent vasodilatation (by 1.2 to 1.6-fold), suppressible by muscarinic and nitric oxide synthase (NOS) antagonists [atropine, NG-nitro-l-arginine methyl ester (l-NAME)] but resistant to nicotinic and serotoninergic antagonists (mecamylamine, methylsergide). Fluoxetine administered 10–30 min. following experimental vascular photo-thrombosis increased arterial diameter (1.3–1.6), inducing partial, but lasting reperfusion of the ischaemic brain. In brain endothelial b.End.3 cells, fluoxetine induced rapid muscarinic receptor-dependent increases in intracellular [Ca2+] and promoted albumin- and eNOS-dependent nitric oxide (NO) production and HSP90 interaction. In vitro, fluoxetine suppressed recombinant human acetylcholinesterase (rhAChE) activity only in the presence of albumin. That fluoxetine induces vasodilatation of cerebral arterioles suggests co-promotion of endothelial muscarinic and nitric oxide signalling, facilitated by albumin-dependent inhibition of serum AChE. PMID:22697296

  10. Extracellular ATP induces albuminuria in pregnant rats

    NARCIS (Netherlands)

    Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

    BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

  11. Lack of correlation between vasodilatation and pharmacologically induced immediate headache in healthy subjects

    DEFF Research Database (Denmark)

    Ashina, Messoud; Tfelt-Hansen, Peer; Dalgaard, Peter

    2011-01-01

    The causal relationship between experimental headache and vasodilatation has not been fully clarified. In the present study, we combined headache and vascular data from eight experimental studies and conducted detailed statistical analyses. Given that substances used in all these experiments were...

  12. Extracellular ATP Induces Calcium Signaling in Odontoblasts.

    Science.gov (United States)

    Lee, B M; Jo, H; Park, G; Kim, Y H; Park, C K; Jung, S J; Chung, G; Oh, S B

    2017-02-01

    Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca(2+) concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca(2+)-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X

  13. Inhibitory effect of chronic oral treatment with fluoxetine on capsaicin-induced external carotid vasodilatation in anaesthetised dogs.

    Science.gov (United States)

    Muñoz-Islas, Enriqueta; González-Hernández, Abimael; Lozano-Cuenca, Jair; Ramírez-Rosas, Martha Beatríz; Medina-Santillán, Roberto; Centurión, David; MaassenVanDenBrink, Antoinette; Villalón, Carlos M

    2015-10-01

    During migraine, capsaicin-sensitive trigeminal sensory nerves release calcitonin gene-related peptide (CGRP), resulting in cranial vasodilatation and central nociception. Moreover, 5-HT is involved in the pathophysiology of migraine and depression. Interestingly, some limited lines of evidence suggest that fluoxetine may be effective in migraine prophylaxis, but the underlying mechanisms are uncertain. Hence, this study investigated the canine external carotid vasodilator responses to capsaicin, α-CGRP and acetylcholine before and after acute and chronic oral treatment with fluoxetine. Forty-eight vagosympathectomised male mongrel dogs were prepared to measure blood pressure, heart rate and external carotid blood flow. The thyroid artery was cannulated for infusions of agonists. In 16 of these dogs, a spinal cannula was inserted (C1-C3) for infusions of 5-HT. The external carotid vasodilator responses to capsaicin, α-CGRP and acetylcholine remained unaffected after intracarotid or i.v. fluoxetine. In contrast, the vasodilator responses to capsaicin, but not those to α-CGRP or acetylcholine, were inhibited after chronic oral treatment with fluoxetine (300 µg/kg; for 90 days) or intrathecal 5-HT. Chronic oral fluoxetine inhibited capsaicin-induced external carotid vasodilatation, and this inhibition could partly explain its potential prophylactic antimigraine action. © International Headache Society 2015.

  14. Acute hyperglycemia and hyperinsulinemia enhance vasodilatation in type 1 diabetes mellitus without increasing capillary permeability and inducing endothelial dysfunction

    NARCIS (Netherlands)

    Kant, GD; Dullaart, RPF; Smit, AJ

    Uncomplicated Type I (insulin-dependent) diabetes mellitus is characterized by generalized vasodilatation. Its possible correlates, increased microvascular permeability and endothelial dysfunction, have been associated with long-term complications. The objective was to study the effects of acute

  15. Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.

    Science.gov (United States)

    Qian, Yanrong; Wang, Xuan; Liu, Yi; Li, Yunsheng; Colvin, Robert A; Tong, Lingying; Wu, Shiyong; Chen, Xiaozhuo

    2014-09-01

    ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

    Science.gov (United States)

    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  17. Alpha-blockade and vasodilatation induced by nipradilol, arotinolol and labetalol in pithed rats.

    Science.gov (United States)

    Nakagawa, Y; Nakahara, H; Chin, W P; Imai, S

    1985-12-01

    In pithed rats two recently-introduced beta-blockers, nipradilol and arotinolol, as well as labetalol shifted the pressor dose-response curve for phenylephrine to the right. Labetalol and arotinolol did not modify the pressor dose-response curve for clonidine, while nipradilol induced a definite rightward shift. These results indicate that labetalol and arotinolol are selective alpha 1-blockers, while nipradilol is a non-selective one. In addition, all the three beta-blockers produced complex changes in the blood pressure in pithed rats. A fall of the diastolic blood pressure induced by labetalol and nipradilol was preceded by a slight rise, while arotinolol produced a fall at lower doses and a rise at higher ones. The hypotension by labetalol was abolished after propranolol, while the hypertension was suppressed by prazosin, indicating that labetalol has an intrinsic beta- and alpha 1-sympathomimetic effect. The hypertension and the hypotension produced by nipradilol and arotinolol persisted even in the presence of propranolol and prazosin or propranolol and yohimbine.

  18. Correction of glycosylated oxyhemoglobin-induced impairment of endothelium-dependent vasodilatation by gliclazide.

    Science.gov (United States)

    Vallejo, S; Angulo, J; Peiró, C; Sánchez-Ferrer, A; Cercas, E; Nevado, J; Sánchez-Ferrer, C F; Rodríguez-Mañas, L

    2000-01-01

    We have investigated whether gliclazide, a second-generation sulfonylurea hypoglycemic agent, interferes with the impairment of endothelium-dependent nitric-oxide-mediated relaxation produced by 14%-glycosylated human oxyhemoglobin (GHHb). For comparative purposes, other agents, like glibenclamide, aminoguanidine, ascorbic acid or superoxide dismutase (SOD), were also tested. GHHb (10 nM) caused a reduction in endothelium-dependent relaxation induced by acetylcholine (1 nM to 10 microM) in both isolated aortic segments and mesenteric microvessels from normoglycemic nondiabetic rats. Preincubation of the vessels with gliclazide (100 nM to 10 microM) prevented the impairment of endothelial relaxation, the threshold concentration of gliclazide being 300 nM. In addition, 10 microM gliclazide also prevented the reduction by 10 nM GHHb of the relaxation induced by exogenous nitric oxide (NO, 10 nM to 100 microM). Determination of superoxide anion release measured by the reduction in ferricytochrome c indicated that GHHb produced significant amounts of these free radicals that were concentration-dependently inhibited by gliclazide. The impairment of endothelium-mediated responses was also prevented by 100 U/ml SOD or 10 microM ascorbic acid, but not by 10 microM glibenclamide or 100 microM aminoguanidine. We conclude that gliclazide can reduce the impairment of nitric-oxide-mediated endothelium-dependent relaxation produced by GHHb. This reduction is likely related to the antioxidant properties of the drug, a mechanism suggested by these studies which demonstrate the inactivation of superoxide anions produced by the glycosylated protein by gliclazide.

  19. Inhibitory effect of BIBN4096BS on cephalic vasodilatation induced by CGRP or transcranial electrical stimulation in the rat

    DEFF Research Database (Denmark)

    Petersen, Kenneth A; Birk, Steffen; Doods, Henri

    2004-01-01

    therapeutic principle. We used an improved closed cranial window model to measure changes of the middle meningeal artery (MMA) and cortical pial artery/arteriole diameter (PA) and changes in local cortical cerebral blood flow (LCBF(Flux)) in anaesthetised artificially ventilated rats. The ability of BIBN4096...... induced by both types of CGRP (P MMA dilatation was reduced from 97.4 +/- 14 to 2.1 +/- 1.3% (P ...CGRP. Transcranial electrical stimulation induced a 119.1 +/- 6.9% increase in MMA diameter. BIBN4096BS (333 microg kg(-1)) attenuated this increase (19.8 +/- 2.1%) (P

  20. Effects of Low versus High Glycemic Index Sugar-Sweetened Beverages on Postprandial Vasodilatation and Inactivity-Induced Impairment of Glucose Metabolism in Healthy Men.

    Science.gov (United States)

    Keller, Judith; Kahlhöfer, Julia; Peter, Andreas; Bosy-Westphal, Anja

    2016-12-10

    Intake of sugar-sweetened beverages (SSB) may contribute to cardiovascular risk. The aim of this study was to investigate whether functional sugars with low compared to high glycemic index (GI) have beneficial effects on arterial stiffness during a period of low-physical activity. In a controlled cross-over dietary intervention (55% CHO, 30% fat, 15% protein), 13 healthy men (age: 23.7 ± 2.2 years, body mass index: 23.6 ± 1.9 kg/m²) completed 2 × 1 week of low physical activity following 1 week of normal physical activity (2363 ± 900 vs. 11,375 ± 3124 steps/day). During inactive phases participants consumed either low-GI (isomaltulose) or high-GI SSB (maltodextrin-sucrose), providing 20% of energy requirements. Postprandial vasodilatation (augmentation index, AIx), insulin sensitivity (IS) and Glucagon-like-peptide 1 (GLP-1) responses were measured during a meal test before and after SSB-intervention. Compared to maltodextrin-sucrose-SSB, postprandial vasodilatation was prolonged (AIx after 120 min: 9.9% ± 4.3% vs. 11.4% ± 3.7%, p 0.05). Higher postprandial GLP-1 secretion after intake of low compared to high-GI beverages may contribute to improved postprandial vasodilatation. Although one week of low-physical activity led to marked impairment in IS, it had no effect on arterial stiffness in healthy men.

  1. Electromagnetic field generation by ATP-induced reverse electron transfer.

    Science.gov (United States)

    Steele, Richard H

    2003-03-01

    This paper describes a mechanism to explain low-level light emission in biology. A biological analog of the electrical circuitry, modeled on the parallel plate capacitor, traversed by a helical structure, required to generate electromagnetic radiation in the optical spectral range, is described. The charge carrier required for the emissions is determined to be an accelerating electron driven by an ATP-induced reverse electron transfer. The radial velocity component, the emission trajectory, of the moving charges traversing helical protein structures in a cyclotron-type mechanism is proposed to be imposed by the ferromagnetic field components of the iron in the iron-sulfur proteins. The redox systems NADH, riboflavin, and chlorophyll were examined with their long-wavelength absorption maxima determining the energetic parameters for the calculations. Potentials calculated from the axial velocity components for the riboflavin and NADH systems were found to equal the standard redox potentials of these systems as measured electrochemically and enzymatically. The mechanics for the three systems determined the magnetic moments, the angular momenta, and the orbital magnetic fluxes to be adiabatic invariant parameters. The De Broglie dual wave-particle equation, the fundamental equation of wave mechanics, and the key idea of quantum mechanics, establishes the wavelengths for accelerating electrons which, divided into a given radial velocity, gives its respective emission frequency. Electrons propelled through helical structures, traversed by biologically available electric and magnetic fields, make accessible to the internal environment the optical spectral frequency range that the solar spectrum provides to the external environment.

  2. FIBROBLAST CYTOSKELETAL REMODELING INDUCED BY TISSUE STRETCH INVOLVES ATP SIGNALING

    OpenAIRE

    Langevin, HM; Fujita, T.; Bouffard, NA; Takano, T; Koptiuch, C; Badger, GJ; Nedergaard, M

    2013-01-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min...

  3. Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling.

    Science.gov (United States)

    Langevin, Helene M; Fujita, Takumi; Bouffard, Nicole A; Takano, Takahiro; Koptiuch, Cathryn; Badger, Gary J; Nedergaard, Maiken

    2013-09-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 min, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediate the active change in fibroblast shape that occurs in response to a static increase in tissue length. Copyright © 2013 Wiley Periodicals, Inc.

  4. TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

    Science.gov (United States)

    Kwon, Hyuck Joon

    2012-08-10

    Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels. This study suggests that Ca(2+)-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation.

    Science.gov (United States)

    Sadatomi, Daichi; Nakashioya, Kazutaka; Mamiya, Sayaka; Honda, Shino; Kameyama, Yuka; Yamamura, Yasuo; Tanimura, Susumu; Takeda, Kohsuke

    2017-06-01

    The NLRP3 inflammasome plays a critical role in the processing and release of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18. Accumulating evidence suggests that mitochondria are common mediators of NLRP3 inflammasome activation induced by a wide range of inflammatory stimuli; however, the precise role of mitochondria is still not fully understood. Here, we show that mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation. Extracellular ATP induced the loss of mitochondrial membrane potential and mitochondrial fragmentation in a different manner than other stimuli in primary mouse macrophages. CCCP, an uncoupler and antimycin A, an inhibitor of the mitochondrial electron transport chain, inhibited IL-1β release induced by ATP but not by other stimuli. CCCP did not inhibit the ATP-induced generation of reactive oxygen species and cell death, both of which are known to promote IL-1β release, but did inhibit the ATP-induced activation of caspase-1, a component of the NLRP3 inflammasome. These results suggest that mitochondrial function is required somewhat specifically for ATP-induced NLRP3 inflammasome activation. In contrast to many previous reports that dysfunctional mitochondria promote NLRP3 inflammasome activation, the function of intact mitochondria appears to be required for NLRP3 inflammasome activation, depending on the stimulus. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

    Science.gov (United States)

    Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

    2010-04-01

    Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

  7. Extracellular ATP modulates synaptic plasticity induced by activation of metabotropic glutamate receptors in the hippocampus.

    Science.gov (United States)

    Yamazaki, Yoshihiko; Fujii, Satoshi

    2015-01-01

    Synaptic plasticity is believed to be a cellular mechanism for memory formation in the brain. It has been known that the metabotropic glutamate receptor (mGluR) is required for persistent forms of memory and induction of synaptic plasticity. Application of mGluR agonists induces synaptic plasticity in the absence of electrical conditioning stimulation, such as high or low frequency stimulation. The direction of the mGluR-induced synaptic plasticity, i.e., either long-term potentiation (LTP) or long-term-depression (LTD), is dependent on whether N-methyl-D-aspartate receptors (NMDARs) are co-activated with mGluRs. ATP has modulatory effects on neuronal functions and, in particular, there is increasing evidence that it plays a crucial role in synaptic plasticity. LTP can be induced by application of ATP, and this effect is inhibited by NMDAR antagonist. Although cooperative effects of NMDARs and mGluRs and of NMDARs and extracellular ATP in synaptic plasticity have been revealed, the effect of extracellular ATP on mGluR-induced synaptic plasticity is unknown. In this article, we summarize published data on mGluR- and ATP-induced synaptic plasticity, and present new data showing that extracellular ATP facilitates both the LTP and LTD induced by mGluR activation.

  8. PYK2 mediates BzATP-induced extracellular matrix proteins synthesis.

    Science.gov (United States)

    Torigoe, Go; Nagao, Mayu; Tanabe, Natsuko; Manaka, Soichiro; Kariya, Taro; Kawato, Takayuki; Sekino, Jumpei; Kato, Shunichiro; Maeno, Masao; Suzuki, Naoto; Shimizu, Noriyoshi

    2017-12-16

    Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2014-01-01

    the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1-10 μM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP...... the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium...

  10. Neurogenic vasodilatation in isolated bovine and canine penile arteries.

    Science.gov (United States)

    Bowman, A; Gillespie, J S

    1983-01-01

    Field stimulation of isolated, perfused bovine or canine penile arteries produced dilatation, after the adrenergic motor component of the response had been blocked with guanethidine and the vessels had developed a background tone. The vasodilatation was blocked by tetrodotoxin but not by atropine. The vasodilator responses to field stimulation were compared with those produced by ATP, by vasoactive intestinal peptide (VIP), and by the inhibitory factor extracted from the bovine retractor penis muscle. Of the three putative transmitters, the inhibitory factor produced responses that most closely resembled those to field stimulation. Haemoglobin, which blocks non-adrenergic, non-cholinergic inhibitory transmission in the bovine and canine retractor penis muscles, did not impair the vasodilatations produced by ATP or VIP, but slowly reduced or abolished those produced by field stimulation or by the inhibitory factor. Haemoglobin itself produced a powerful constriction of the isolated penile arteries. The results are compatable with the possibility that the inhibitory factor from the bovine retractor penis muscle (which may be the inhibitory transmitter in that muscle) is, or closely resembles, the transmitter of non-adrenergic, non-cholinergic vasodilator fibres in the penile arteries of dog and ox. PMID:6684686

  11. Continuous detection of cerebral vasodilatation and vasoconstriction using intracranial pulse morphological template matching.

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    Shadnaz Asgari

    Full Text Available Although accurate and continuous assessment of cerebral vasculature status is highly desirable for managing cerebral vascular diseases, no such method exists for current clinical practice. The present work introduces a novel method for real-time detection of cerebral vasodilatation and vasoconstriction using pulse morphological template matching. Templates consisting of morphological metrics of cerebral blood flow velocity (CBFV pulse, measured at middle cerebral artery using Transcranial Doppler, are obtained by applying a morphological clustering and analysis of intracranial pulse algorithm to the data collected during induced vasodilatation and vasoconstriction in a controlled setting. These templates were then employed to define a vasodilatation index (VDI and a vasoconstriction index (VCI for any inquiry data segment as the percentage of the metrics demonstrating a trend consistent with those obtained from the training dataset. The validation of the proposed method on a dataset of CBFV signals of 27 healthy subjects, collected with a similar protocol as that of training dataset, during hyperventilation (and CO₂ rebreathing tests shows a sensitivity of 92% (and 82% for detection of vasodilatation (and vasoconstriction and the specificity of 90% (and 92%, respectively. Moreover, the proposed method of detection of vasodilatation (vasoconstriction is capable of rejecting all the cases associated with vasoconstriction (vasodilatation and outperforms other two conventional techniques by at least 7% for vasodilatation and 19% for vasoconstriction.

  12. Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells

    Science.gov (United States)

    Koyama, Tetsuya; Oike, Masahiro; Ito, Yushi

    2001-01-01

    Hypotonic stress induces ATP release followed by Ca2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca2+ oscillations induced by hypotonic stress. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca2+ oscillations, indicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic ATP release. Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca2+ oscillations. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs. PMID:11313444

  13. Retinal Changes in an ATP-Induced Model of Retinal Degeneration

    Science.gov (United States)

    Aplin, Felix P.; Vessey, Kirstan A.; Luu, Chi D.; Guymer, Robyn H.; Shepherd, Robert K.; Fletcher, Erica L.

    2016-01-01

    In rodents and felines, intravitreal administration of adenosine triphosphate (ATP) has been shown to induce photoreceptor death providing a tractable model of retinal degeneration in these species. This study investigated the long term effects of photoreceptor loss in an ATP induced feline model of retinal degeneration. Six normal sighted felines were unilaterally blinded using intravitreal ATP injections and assessed using electroretinography (ERG) and optical coherence tomography (OCT). At 30 h (n = 3) or 12 weeks (n = 3) post-injection, the animals were euthanized and the eyes enucleated. Retinae were sectioned and labeled using immunohistochemistry for markers of cell death, neural remodeling and gliosis. Ongoing cell death and retinal degeneration was observed in the outer retina at both 30 h and 12 weeks following unilateral ATP injection. Markers of mid to late-stage retinal remodeling such as cell displacement and aberrant neurite growth were observed in the inner retina at 12 weeks post-injection. Ganglion cells appeared to remain intact in ATP injected eyes. Müller cell gliosis was observed throughout the inner and outer retina, in some parts completely enveloping and/or displacing the surviving neural tissue. Our data suggests that the ATP injected feline retina continues to undergo progressive retinal degeneration and exhibits abnormalities consistent with a description of retinal remodeling commonly seen in other models of retinal degeneration. These findings validate the use of intravitreal ATP injection in feline as a large animal model of retinal degeneration which may aid in development of therapies aiming to restore visual function after photoreceptor degeneration. PMID:27199678

  14. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

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    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  15. Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion.

    Science.gov (United States)

    Tong, Jin; Zhou, Xiang-Dong; Perelman, Juliy M; Kolosov, Victor P

    2014-02-01

    The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA-AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT-PCR (reverse transcription-PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA-AM and RB-2, while EGTA did not have a suppressive effect. BAPTA-AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca(2+)-dependent ATP release, and nearly all Ca(2+) was provided by stored intracellular Ca(2). © 2014 The author(s).

  16. Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion

    Science.gov (United States)

    Tong, Jin; Zhou, Xiang-dong; Perelman, Juliy M.; Kolosov, Victor P.

    2013-01-01

    The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA–AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid–acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT–PCR (reverse transcription–PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA–AM and RB-2, while EGTA did not have a suppressive effect. BAPTA–AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+-dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+. PMID:27919041

  17. Correlated ATP-induced changes in membrane area and membrane conductance in single rat adipocytes.

    Science.gov (United States)

    Chowdhury, Helena H; Grilc, Sonja; Zorec, Robert

    2005-06-01

    In the past few years it has been shown that, like many other non-neuroendocrine cells, adipocytes possess a mechanism for triggered exocytosis. Endocytosis and exocytosis affect the plasma membrane surface area, which can be directly monitored with electrophysiological patch-clamp techniques by measuring membrane capacitance, a parameter linearly related to the plasma membrane area. In this study we used the whole-cell mode of the patch-clamp technique to measure changes in membrane capacitance to monitor the effect of extracellular adenosine triphosphate (ATP) on the dynamics of membrane area changes in single adipocytes. Experimental evidence shows that extracellular application of ATP (100 microM) increases membrane capacitance for 30 +/- 2%. In controls a significantly smaller increase of 3 +/- 2% was measured, which is due to a slow exocytic-endocytic membrane cycling rate of 0.3%/min. We found that ATP induces a transient increase in membrane current, temporally associated with the peak rate in membrane capacitance increase. These results show directly the presence of ATP-induced increase in membrane area correlated to the increase in membrane current in single adipocytes.

  18. Impact of age on exercise-induced ATP supply during supramaximal plantar flexion in humans.

    Science.gov (United States)

    Layec, Gwenael; Trinity, Joel D; Hart, Corey R; Kim, Seong-Eun; Groot, H Jonathan; Le Fur, Yann; Sorensen, Jacob R; Jeong, Eun-Kee; Richardson, Russell S

    2015-08-15

    Currently, the physiological factors responsible for exercise intolerance and bioenergetic alterations with age are poorly understood due, at least in art, to the confounding effect of reduced physical activity in the elderly. Thus, in 40 healthy young (22 ± 2 yr) and old (74 ± 8 yr) activity-matched subjects, we assessed the impact of age on: 1) the relative contribution of the three major pathways of ATP synthesis (oxidative ATP synthesis, glycolysis, and the creatine kinase reaction) and 2) the ATP cost of contraction during high-intensity exercise. Specifically, during supramaximal plantar flexion (120% of maximal aerobic power), to stress the functional limits of the skeletal muscle energy systems, we used (31)P-labeled magnetic resonance spectroscopy to assess metabolism. Although glycolytic activation was delayed in the old, ATP synthesis from the main energy pathways was not significantly different between groups. Similarly, the inferred peak rate of mitochondrial ATP synthesis was not significantly different between the young (25 ± 8 mM/min) and old (24 ± 6 mM/min). In contrast, the ATP cost of contraction was significantly elevated in the old compared with the young (5.1 ± 2.0 and 3.7 ± 1.7 mM·min(-1)·W(-1), respectively; P age-related mitochondrial and glycolytic dysfunction. However, this study does confirm an abnormal elevation in exercise-induced skeletal muscle metabolic demand in the old that may contribute to the decline in exercise capacity with advancing age.

  19. Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia.

    Science.gov (United States)

    Alberici, Luciane C; Paim, Bruno A; Zecchin, Karina G; Mirandola, Sandra R; Pestana, Cezar R; Castilho, Roger F; Vercesi, Anibal E; Oliveira, Helena C F

    2013-06-14

    We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoK(ATP) channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

  20. Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition

    Science.gov (United States)

    Park, Dohyun; Jeong, Heeyoon; Lee, Mi Nam; Koh, Ara; Kwon, Ohman; Yang, Yong Ryoul; Noh, Jungeun; Suh, Pann-Ghill; Park, Hwangseo; Ryu, Sung Ho

    2016-01-01

    Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction. PMID:26902888

  1. Distinct cell stress responses induced by ATP restriction in quiescent human fibroblasts

    Directory of Open Access Journals (Sweden)

    Nirupama Yalamanchili

    2016-10-01

    Full Text Available Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

  2. SIRT2 and Akt mediate NAD+-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia under basal conditions.

    Science.gov (United States)

    Zhang, Jie; Wang, Caixia; Ying, Weihai

    2018-01-17

    NAD replenishment can restore ATP levels and rescue premature aging in Cockayne syndrome mice. However, there has been no mechanistic study regarding the effects of NAD and NADH on intracellular ATP levels under basal conditions. In our current study, we used BV2 microglia to test our hypothesis that NAD and NADH can increase intracellular ATP levels under basal conditions. We found that both NAD and NADH significantly increased the intracellular ATP levels of BV2 microglia, which were attenuated by SIRT2 siRNA, the SIRT2 inhibitor AGK2, and the phosphatidylinositol 3-kinase/Akt inhibitor LY294002. Our study has also suggested that SIRT2 mediates the NAD-induced and NADH-induced increase in Akt phosphorylation in BV2 microglia. Collectively, our study has suggested that SIRT2 mediates both NAD-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia by modulating Akt phosphorylation.

  3. ATP-induced inflammasome activation and pyroptosis is regulated by AMP-activated protein kinase in macrophages

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    Qing-Bing Zha

    2016-12-01

    Full Text Available ATP is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist. Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation.

  4. ATP induces NO production in hippocampal neurons by P2X(7 receptor activation independent of glutamate signaling.

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    Juan Francisco Codocedo

    Full Text Available To assess the putative role of adenosine triphosphate (ATP upon nitric oxide (NO production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3'-O-(4-Benzoylbenzoyl ATP (Bz-ATP elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG or by N(ω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV, but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

  5. CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

    Science.gov (United States)

    Long, Jingdong; Lei, Xiaolu; Chen, Meiyun; Yang, Shulei; Sun, Tao; Zeng, Junwei; Yu, Deqian; Tian, Hong; Liu, Xiaohong

    2017-11-10

    Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening

  6. Intractable hyperkalemia due to nicorandil induced potassium channel syndrome

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    Vivek Chowdhry

    2015-01-01

    Full Text Available Nicorandil is a commonly used antianginal agent, which has both nitrate-like and ATP-sensitive potassium (K ATP channel activator properties. Activation of potassium channels by nicorandil causes expulsion of potassium ions into the extracellular space leading to membrane hyperpolarization, closure of voltage-gated calcium channels and finally vasodilatation. However, on the other hand, being an activator of K ATP channel, it can expel K + ions out of the cells and can cause hyperkalemia. Here, we report a case of nicorandil induced hyperkalemia unresponsive to medical treatment in a patient with diabetic nephropathy.

  7. F1F0 ATP Synthase–Cyclophilin D Interaction Contributes to Diabetes-Induced Synaptic Dysfunction and Cognitive Decline

    Science.gov (United States)

    Yan, Shijun; Du, Fang; Wu, Long; Zhang, Zhihua; Zhong, Changjia; Yu, Qing; Wang, Yongfu; Lue, Lih-Fen; Walker, Douglas G.; Douglas, Justin T.

    2016-01-01

    Mitochondrial abnormalities are well known to cause cognitive decline. However, the underlying molecular basis of mitochondria-associated neuronal and synaptic dysfunction in the diabetic brain remains unclear. Here, using a mitochondrial single-channel patch clamp and cyclophilin D (CypD)-deficient mice (Ppif −/−) with streptozotocin-induced diabetes, we observed an increase in the probability of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in brain mitochondria of diabetic mice, which was further confirmed by mitochondrial swelling and cytochrome c release induced by Ca2+ overload. Diabetes-induced elevation of CypD triggers enhancement of F1F0 ATP synthase–CypD interaction, which in turn leads to mPTP opening. Indeed, in patients with diabetes, brain cypD protein levels were increased. Notably, blockade of the F1F0 ATP synthase–CypD interaction by CypD ablation protected against diabetes-induced mPTP opening, ATP synthesis deficits, oxidative stress, and mitochondria dysfunction. Furthermore, the absence of CypD alleviated deficits in synaptic plasticity, learning, and memory in diabetic mice. Thus, blockade of ATP synthase interaction with CypD provides a promising new target for therapeutic intervention in diabetic encephalopathy. PMID:27554467

  8. Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation*

    Science.gov (United States)

    Pinheiro, Ana Rita; Paramos-de-Carvalho, Diogo; Certal, Mariana; Costa, Maria Adelina; Costa, Cristina; Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Sévigny, Jean; Correia-de-Sá, Paulo

    2013-01-01

    Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors. PMID:23918924

  9. Involvement of the PI3K/AKT pathway in ATP-induced proliferation of developing retinal cells in culture.

    Science.gov (United States)

    Ornelas, Isis Moraes; Ventura, Ana Lucia Marques

    2010-10-01

    ATP induces the proliferation of chick retinal cells in culture through the activation of P2Y1 receptors, PKC and MAP kinases. Together with MAP kinases, the PI3K/AKT pathway has also been implicated as an important mediator in proliferative events during development. Here we investigated the participation of the PI3K/AKT signal pathway on ATP-induced proliferation of chick embryo retinal cells in culture. When retinal cultures obtained from 7-day-old embryos were cultivated for 1 day and treated with ATP, a transient and dose-dependent phosphorylation of both ERK and AKT was observed, an effect that could be mimicked by 500 microM ADP and blocked by 100 microM PPADS, a P2 receptor antagonist. Maximal stimulation of both enzymes was obtained with 100 microM ATP in 5 min, decreasing thereafter. Activation of these pathways by ATP seemed to be independent, since LY294002 and U0126, inhibitors of PI3K and MEK, did not block the activation of ERK and AKT, respectively, although each compound blocked its respective target. Moreover, when the cultures were incubated with ATP in the presence of LY294002, a decreased incorporation of [(3)H]-thymidine was observed, as compared to cultures treated only with ATP, a decline that was also obtained by incubating the cells with ATP plus 0.5 microM API-59CJ-Ome, an inhibitor of AKT. No decrease in cell viability was observed with this concentration of API-59CJ-Ome. An increase in cyclin D1 expression, that could be inhibited by 10 microM LY 294002 or 20 microM U0126, was observed when cells were incubated with 500 microM ADP. No effect of PI3K and MEK inhibitors was observed in the expression of p27kip1 in the cultures. These results suggest that, besides the involvement of the MAP kinases pathway, ATP-induced cell cycling of late developing retinal progenitors in culture also involves the activation of the PI3K/AKT pathway. Copyright 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

  10. Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium.

    Science.gov (United States)

    Into, Takeshi; Okada, Kazutaka; Inoue, Nobuo; Yasuda, Motoaki; Shibata, Ken-ichiro

    2002-01-01

    The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.

  11. The pathophysiology of arterial vasodilatation and hyperdynamic circulation in cirrhosis

    DEFF Research Database (Denmark)

    Møller, Søren; Bendtsen, Flemming

    2017-01-01

    transplantation and point to the pathophysiological significance of portal hypertension. In this paper we aimed to review current knowledge on the pathophysiology of arterial vasodilatation and the hyperdynamic circulation in cirrhosis. This article is protected by copyright. All rights reserved....

  12. Phosphorylation of β-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells

    Science.gov (United States)

    Taurin, Sebastien; Sandbo, Nathan; Yau, Douglas M.; Sethakorn, Nan; Dulin, Nickolai O.

    2012-01-01

    Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449–C456, 2004). We also have shown that PKA can phosphorylate β-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971–9976, 2006). β-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of β-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous β-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) β-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous β-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC. PMID:18353896

  13. Phosphorylation of beta-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells.

    Science.gov (United States)

    Taurin, Sebastien; Sandbo, Nathan; Yau, Douglas M; Sethakorn, Nan; Dulin, Nickolai O

    2008-05-01

    Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.

  14. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

    Science.gov (United States)

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-06-23

    HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

  15. ATP-Sensitive K+ Channel Knockout Induces Cardiac Proteome Remodeling Predictive of Heart Disease Susceptibility

    Science.gov (United States)

    Arrell, D. Kent; Zlatkovic, Jelena; Kane, Garvan C.; Yamada, Satsuki; Terzic, Andre

    2010-01-01

    Forecasting disease susceptibility requires detection of maladaptive signatures prior to onset of overt symptoms. A case-in-point are cardiac ATP-sensitive K+ (KATP) channelopathies, for which the substrate underlying disease vulnerability remains to be identified. Resolving molecular pathobiology, even for single genetic defects, mandates a systems platform to reliably diagnose disease predisposition. High-throughput proteomic analysis was here integrated with network biology to decode consequences of Kir6.2 KATP channel pore deletion. Differential two-dimensional gel electrophoresis reproducibly resolved > 800 protein species from hearts of asymptomatic wild-type and Kir6.2-knockout counterparts. KATP channel ablation remodeled the cardiac proteome, significantly altering 71 protein spots, from which 102 unique identities were assigned following hybrid linear ion trap quadrupole-Orbitrap tandem mass spectrometry. Ontological annotation stratified the KATP channel-dependent protein cohort into a predominant bioenergetic module (63 resolved identities), with additional focused sets representing signaling molecules (6), oxidoreductases (8), chaperones (6), and proteins involved in catabolism (6), cytostructure (8), and transcription and translation (5). Protein interaction mapping, in conjunction with expression level changes, localized a KATP channel-associated subproteome within a nonstochastic scale-free network. Global assessment of the KATP channel deficient environment verified the primary impact on metabolic pathways and revealed overrepresentation of markers associated with cardiovascular disease. Experimental imposition of graded stress precipitated exaggerated structural and functional myocardial defects in the Kir6.2-knockout, decreasing survivorship and validating the forecast of disease susceptibility. Proteomic cartography thus provides an integral view of molecular remodeling in the heart induced by KATP channel deletion, establishing a systems

  16. Dynamic changes in cytosolic ATP levels in cultured glutamatergic neurons during NMDA-induced synaptic activity supported by glucose or lactate

    DEFF Research Database (Denmark)

    Lange, Sofie Cecilie; Winkler, Ulrike; Andresen, Lars

    2015-01-01

    We have previously shown that synaptic transmission fails in cultured neurons in the presence of lactate as the sole substrate. Thus, to test the hypothesis that the failure of synaptic transmission is a consequence of insufficient energy supply, ATP levels were monitored employing the ATP...... biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30 s pulses of NMDA (30 µM) with a 4 min interval, changes in relative ATP levels were measured in the presence of lactate (1 mM), glucose (2.5 mM) or the combination of the two. ATP levels reversibly declined...... following NMDA-induced neurotransmission activity, as indicated by a reversible 10-20 % decrease in the response of the biosensor. The responses were absent when the NMDA receptor antagonist memantine was present. In the presence of lactate alone, the ATP response dropped significantly more than...

  17. Endothelial ATP-binding cassette G1 in mouse endothelium protects against hemodynamic-induced atherosclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Shanshan [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Pediatrics, Baodi District People’s Hospital of Tianjin City, Tianjin, 301800 (China); Wang, Jiaxing [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Zhang, Xu; Shi, Ying; Li, Bochuan; Bao, Qiankun [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Pang, Wei [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Ai, Ding [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Zhu, Yi [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); He, Jinlong, E-mail: hejinlong@tmu.edu.cn [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China)

    2016-08-19

    Activated vascular endothelium inflammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid homeostasis by transporting cholesterol efflux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inflammation activation during early-stage atherogenesis in mice and the underlying mechanisms. Endothelial cell (EC)-specific ABCG1 transgenic (EC-ABCG1-Tg) mice were generated and cross-bred with low-density lipoprotein receptor–deficient (Ldlr{sup −/−}) mice. After a 4-week Western-type diet, the mice were sacrificed for assessing atherosclerosis. Human umbilical vein ECs were treated with different flows, and ABCG1 was adenovirally overexpressed to investigate the mechanism in vitro. Compared with Ldlr{sup −/−} mouse aortas, EC-ABCG1-Tg/Ldlr{sup −/−} aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr{sup −/−} mouse aortic arch but not thoracic aorta was significantly reduced, which suggests a protective role of ABCG1 under atheroprone flow. In vitro, overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. Overexpression of ABCG1 blunted cholesterol-activated ECs in vitro. In exploring the mechanisms of ABCG1 attenuating endothelial inflammation, we found that ABCG1 inhibited oscillatory flow-activated nuclear factor kappa B and NLRP3 inflammasome in ECs. ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory shear stress via suppressing the inflammatory response. - Highlights: • EC-ABCG1-Tg mice in a Ldlr{sup −/−} background showed decreased atherosclerosis. • Overexpression of ABCG1 in ECs decreased OSS-induced EC activation. • NLRP3 and NF-κB might be an underlying mechanism of ABCG1 protective role.

  18. Comparison of ATP-MgCl2 and Methylprednisolone in Experimentally Induced Spinal Cord Trauma

    Directory of Open Access Journals (Sweden)

    Yusuf Şirin

    2012-10-01

    Full Text Available Aim: The aim of this study is to compare the preventive effects of methyl prednisolone sodium succinate (MPSS and ATP-MgCl2 on secondary injury after spinal cord trauma by clinical, electrophysiological and histopathological examination. Material and Method: Thirty healthy male Wistar-Albino rats were used in the study. All rats were evaluated by inclined plane, toe-spread and tarlov tests. Before the operation, somatosensoric evoked potentials (SEP and motor evoked potentials (MEP were recorded from anesthetized rats. The spinal cord was traumatised by Allen’s weight drop method applying 50gr/cm force. The rats were divided into 3 groups of 10 members per group (1st group FTS, 2nd group-MP, 3rd group ATP. The first group received saline, the second group received MPSS, and the third group received ATP-MgCl2. At regular intervals after the operation, these tests were repeated on the rats. At the 72nd hour after operation, their SEP and MEP values were recorded again. The damaged spinal cord segment stained with hematoxylin - eosin was evaluated histopathologically. Results: Qualitative SEP scales and Tarlov scales were found compatible. MP and ATP groups were found significantly better than control group (FTS according to some electrophysiological results. However, a significant difference could not be determined between MPSS and ATP-MgCl2 applied groups. Histopathological examinations showed the presence of MPSS caused polymorphonuclear cell reaction. Discussion: As a result, more experimental studies are needed to say that ATP-MgCl2 is more effective than MPSS in spinal cord injury (SCI.

  19. ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct

    DEFF Research Database (Denmark)

    Svenningsen, Per; Burford, James L; Peti-Peterdi, János

    2013-01-01

    in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30...

  20. Breast feeding increases vasoconstriction induced by electrical field stimulation in rat mesenteric artery. Role of neuronal nitric oxide and ATP.

    Directory of Open Access Journals (Sweden)

    Javier Blanco-Rivero

    Full Text Available OBJECTIVES: The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF affects the vasomotor response induced by electrical field stimulation (EFS, participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications. METHODS: Experiments were performed in female Sprague-Dawley rats (3 months old, divided into three groups: Control (in oestrous phase, mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase. Vasomotor response to EFS, noradrenaline (NA and nitric oxide (NO donor DEA-NO were studied. Neuronal NO synthase (nNOS and phosphorylated nNOS (P-nNOS protein expression were analysed and NO, superoxide anion (O(2(.-, NA and ATP releases were also determined. RESULTS: EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O(2(.- production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals. CONCLUSION: Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.

  1. Quebrachitol-induced gastroprotection against acute gastric lesions: role of prostaglandins, nitric oxide and K+ ATP channels.

    Science.gov (United States)

    de Olinda, T M; Lemos, T L G; Machado, L L; Rao, V S; Santos, F A

    2008-05-01

    The effect of Quebrachitol (2-O-methyl-L-inositol), a bioactive component from Magonia glabrata fruit extract was investigated against gastric damage induced by absolute ethanol (96%, 0.2 ml/animal) and indomethacin (30 mg/kg, p.o.), in mice. Quebrachitol at oral doses of 12.5, 25, and 50mg/kg markedly attenuated the gastric lesions induced by ethanol to the extent of 69%, 64%, and 53% and against indomethacin by 55%, 59%, and 26%, respectively. While pretreatment with TRPV1 antagonist capsazepine (5mg/kg, i.p.) failed to block effectively the gastroprotective effect of quebrachitol (25mg/kg) against ethanol damage, the non-selective cyclooxygenase inhibitor indomethacin (10mg/kg, p.o.), almost abolished it. Furthermore, quebrachitol effect was significantly reduced in mice pretreated with L-NAME, or glibenclamide, the respective inhibitors of nitric oxide synthase and K(+)(ATP) channel activation. Thus we provide the first evidence that quebrachitol reduces the gastric damage induced by ethanol and indomethacin, at least in part, by mechanisms that involve endogenous prostaglandins, nitric oxide release, and or the activation of K(+)(ATP) channels.

  2. Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

    Directory of Open Access Journals (Sweden)

    Hey-Kyeong Jeong

    Full Text Available BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+ and Iba-1(+ cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular

  3. Epigenetic switch at atp2a2 and myh7 gene promoters in pressure overload-induced heart failure.

    Directory of Open Access Journals (Sweden)

    Tiziana Angrisano

    Full Text Available Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF. Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca2+ATPase (SERCA-2A and β-myosin-heavy chain (β-MHC genes (Atp2a2 and Myh7, respectively in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC. As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2 at lysine (K 4 (H3K4me2 and K9 (H3K9me2, H3 trimethylation (me3 at K27 (H3K27me3 and dimethylation (me2 at K36 (H3K36me2. Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2 were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.

  4. Epigenetic switch at atp2a2 and myh7 gene promoters in pressure overload-induced heart failure.

    Science.gov (United States)

    Angrisano, Tiziana; Schiattarella, Gabriele Giacomo; Keller, Simona; Pironti, Gianluigi; Florio, Ermanno; Magliulo, Fabio; Bottino, Roberta; Pero, Raffaela; Lembo, Francesca; Avvedimento, Enrico Vittorio; Esposito, Giovanni; Trimarco, Bruno; Chiariotti, Lorenzo; Perrino, Cinzia

    2014-01-01

    Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca2+ATPase (SERCA-2A) and β-myosin-heavy chain (β-MHC) genes (Atp2a2 and Myh7, respectively) in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC). As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of β-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2) were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.

  5. ATP-induced temperature independence of hemoglobin-O2 affinity in heterothermic billfish

    DEFF Research Database (Denmark)

    Weber, Roy E.; Campbell, Kevin L.; Fago, Angela

    2010-01-01

    heterotherms, where it may hamper unloading (e.g. in cold extremities of arctic mammals) or increase the diffusive arterio-venous short-circuiting of O2 (e.g. in counter-current heat exchangers of warm swimming muscles of tuna). We hypothesized analogous blood specializations in heterothermic billfish, whose...... to allosterically modulating hemoglobin-O2 affinity, ATP diminishes its temperature sensitivity, reducing deleterious arterio-venous short-circuiting of oxygen in the cranial billfish heat exchangers. The mechanism underlying this reduction in oxygenation enthalpy differs fundamentally from that in tuna, supporting...

  6. ATP-induced temperature independence of hemoglobin-O2 affinity in heterothermic billfish

    DEFF Research Database (Denmark)

    Weber, Roy E.; Campbell, Kevin L.; Fago, Angela

    2010-01-01

    heterotherms, where it may hamper unloading (e.g. in cold extremities of arctic mammals) or increase the diffusive arterio-venous short-circuiting of O2 (e.g. in counter-current heat exchangers of warm swimming muscles of tuna). We hypothesized analogous blood specializations in heterothermic billfish, whose...... to allosterically modulating hemoglobin-O2 affinity, ATP diminishes its temperature sensitivity, reducing deleterious arterio venous short-circuiting of oxygen in the cranial billfish heat exchangers. The mechanism underlying this reduction in oxygenation enthalpy differs fundamentally from that in tuna, supporting...

  7. Vildagliptin improves endothelium-dependent vasodilatation in type 2 diabetes

    NARCIS (Netherlands)

    Poppel, P.C. van; Netea, M.G.; Smits, P.; Tack, C.J.J.

    2011-01-01

    OBJECTIVE: To investigate whether the dipeptidyl peptidase-4 inhibitor vildagliptin improves endothelium-dependent vasodilatation in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Sixteen subjects with type 2 diabetes (age 59.8 +/- 6.8 years, BMI 29.1 +/- 4.8 kg/m(2), HbA(1c) 6.97 +/-

  8. ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

    Directory of Open Access Journals (Sweden)

    Per eSvenningsen

    2013-10-01

    Full Text Available ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC and AQP2. Recently, we have shown that connexin (Cx 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30-/- mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i signaling in the CD. Cortical CDs (CCDs from wild type and Cx30-/- mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30-/- CCDs ([Ca2+]i increased only 1.2-fold, p

  9. Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy [IIT; (Penn)

    2013-12-10

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

  10. Muscle α-adrenergic responsiveness during exercise and ATP-induced vasodilation in chronic obstructive pulmonary disease patients

    DEFF Research Database (Denmark)

    Iepsen, Ulrik Winning; Munch, Gregers Druedal Wibe; Ryrsø, Camilla Koch

    2017-01-01

    <0.05) at 20% WLmax in the COPD patients. Tyramine reduced LVC in both groups at 10 W exercise (COPD: -3 ±1; controls: -3±1 mL min-1mmHg-1 P<0.05, respectively) and 20% WLmax (COPD: -4±1; controls: -3±1 mL min-1mmHg-1 P<0.05, respectively) with no difference between groups. Incremental ATP...... infusions induced dose-dependent vasodilation with no difference between groups and also the vasoconstrictor response to Tyramine infused together with ATP was not different between groups (COPD: -0.03±0.01 versus controls: -0.04±0.01 L min-1 kg leg mass-1, P>0.05). Compared with age-matched healthy......Sympathetic vasoconstriction is blunted in exercising muscle (functional sympatholysis) but becomes attenuated with age. We tested the hypothesis that functional sympatholysis is further impaired in chronic obstructive pulmonary disease (COPD) patients. We determined leg blood flow (LBF...

  11. Structure of dimeric ATP synthase from mitochondria : An angular association of monomers induces the strong curvature of the inner membrane

    NARCIS (Netherlands)

    Dudkina, Natalya V.; Heinemeyer, Jesco; Keegstra, Wilko; Boekema, Egbert J.; Braun, Hans-Peter

    2005-01-01

    Respiration in all cells depends upon synthesis of ATP by the ATP synthase complex, a rotary motor enzyme. The structure of the catalytic moiety of ATP synthase, the so-called F1 headpiece, is well established. F1 is connected to the membrane-bound and ion translocating F0 subcomplex by a central

  12. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  13. IL-1ra Secreted by ATP-Induced P2Y2 Negatively Regulates MUC5AC Overproduction via PLCβ3 during Airway Inflammation

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    Jee-Yeong Jeong

    2016-01-01

    Full Text Available Mucus secretion is often uncontrolled in many airway inflammatory diseases of humans. Identifying the regulatory pathway(s of mucus gene expression, mucus overproduction, and hypersecretion is important to alleviate airway inflammation in these diseases. However, the regulatory signaling pathway controlling mucus overproduction has not been fully identified yet. In this study, we report that the ATP/P2Y2 complex secretes many cytokines and chemokines to regulate airway inflammation, among which IL-1 receptor antagonist (IL-1ra downregulates MUC5AC gene expression via the inhibition of Gαq-induced Ca2+ signaling. IL-1ra inhibited IL-1α protein expression and secretion, and vice versa. Interestingly, ATP/P2Y2-induced IL-1ra and IL-1α secretion were both mediated by PLCβ3. A dominant-negative mutation in the PDZ-binding domain of PLCβ3 inhibited ATP/P2Y2-induced IL-1ra and IL-1α secretion. IL-1α in the presence of the ATP/P2Y2 complex activated the ERK1/2 pathway in a greater degree and for a longer duration than the ATP/P2Y2 complex itself, which was dramatically inhibited by IL-1ra. These findings suggest that secreted IL-1ra exhibits a regulatory effect on ATP/P2Y2-induced MUC5AC gene expression, through inhibition of IL-1α secretion, to maintain the mucus homeostasis in the airway. Therefore, IL-1ra could be an excellent modality for regulating inflamed airway microenvironments in respiratory diseases.

  14. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa, E-mail: nebojsa.andric@dbe.uns.ac.rs

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  15. Inhibitory effect of BIBN4096BS, CGRP(8-37), a CGRP antibody and an RNA-Spiegelmer on CGRP induced vasodilatation in the perfused and non-perfused rat middle cerebral artery

    DEFF Research Database (Denmark)

    Edvinsson, L; Nilsson, E; Jansen-Olesen, I

    2007-01-01

    experiments, relaxation induced by alphaCGRP was prevented by the four CGRP blockers (CGRP8-37, BIBN4096BS, the CGRP antibody and NOX-C89.). In abluminal perfusion experiments, the relaxant response to alphaCGRP was prevented by these agents to a varying degree. Dilatation induced by abluminal application...... of alphaCGRP was inhibited by luminal CGRP8-37 but not by luminal BIBN4096BS, CGRP antibody or NOX-C89. CONCLUSIONS AND IMPLICATIONS: alpha or betaCGRP acted on smooth muscle cell CGRP receptors in rat MCA and were effectively prevented from reaching these receptors by the endothelial barrier. The CGRP...

  16. A role for ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion.

    Science.gov (United States)

    Guay, Claudiane; Madiraju, S R Murthy; Aumais, Alexandre; Joly, Erik; Prentki, Marc

    2007-12-07

    In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.

  17. ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis

    Directory of Open Access Journals (Sweden)

    Killen García

    2016-01-01

    Full Text Available Neisseria gonorrhoeae (Ngo has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM. Here, we investigate the role of adenosine triphosphate (ATP in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P0.05 and caspase-1 (CASP1, P>0.05. In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P>0.01. Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

  18. Local anesthetics induce a decrease in the levels of glucose 1, 6-bisphosphate, fructose 1,6-bisphosphate, and ATP, and in the viability of melanoma cells.

    Science.gov (United States)

    Karniel, M; Beitner, R

    2000-01-01

    Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of local anesthetics, lidocaine and bupivacaine, on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP levels and cell viability in B16 melanoma cells. We found that both drugs induced a significant, dose-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1, 6-bisphosphate, ATP, and cell viability. Bupivacaine was more potent than lidocaine. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, induced by the local anesthetics, preceded the reduction in the viability of melanoma cells, indicating that these are early changes and not a result of cell death. Cell viability was reduced in a close correlation with the fall in ATP. These findings suggest that the fall in the levels of the two signal allosteric regulators of glycolysis, induced by the local anesthetics, is one of the mechanisms that causes a reduction in glycolysis and ATP levels, which eventually leads to melanoma cell death. These experiments suggest that local anesthetics, and especially bupivacaine, are most promising agents in the treatment of melanoma. Copyright 2000 Academic Press.

  19. Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion

    NARCIS (Netherlands)

    Roelofsen, H; Wolters, H; Van Luyn, MJA; Miura, N; Kuipers, F; Vonk, RJ

    Background & Aims: Mutations in the ATP7B gene, encoding a copper-transporting P-type adenosine triphosphatase, lead to excessive hepatic copper accumulation because of impaired biliary copper excretion in Wilson's disease. In human liver, ATP7B is predominantly localized to the trans-Golgi network,

  20. ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro

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    Fuxing Yang

    2016-01-01

    Full Text Available Disruption of blood-brain barrier (BBB follows brain trauma or central nervous system (CNS stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R by ATP induces the release of interleukin-1β (IL-1β, which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9. Degradation of tight junction proteins (TJPs such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3′-O-(4-benzoylbenzoyl adenosine 5′-triphosphate (BzATP, induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

  1. Defects in beta cell Ca²+ signalling, glucose metabolism and insulin secretion in a murine model of K(ATP) channel-induced neonatal diabetes mellitus.

    Science.gov (United States)

    Benninger, R K P; Remedi, M S; Head, W S; Ustione, A; Piston, D W; Nichols, C G

    2011-05-01

    Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose

  2. Sildenafil citrate (Viagra) enhances vasodilatation in fetal growth restriction.

    Science.gov (United States)

    Wareing, Mark; Myers, Jenny E; O'Hara, Maureen; Baker, Philip N

    2005-05-01

    Fetal growth restriction (FGR) affects up to 8% of all pregnancies and has massive short-term (increased fetal morbidity and mortality) and long-term (increased incidence of cardiovascular disease in adulthood) health implications. Doppler waveform analysis of pregnancies complicated by FGR suggests compromised uteroplacental circulation and placental hypoperfusion. Our aim was to determine whether myometrial small artery function was aberrant in FGR and to assess whether sildenafil citrate could improve vasodilatation in FGR pregnancies. Small arteries dissected from myometrial biopsies obtained at cesarean section from normal pregnant women (n = 27) or women whose pregnancies were complicated by FGR (n = 12) were mounted on wire myographs. Vessels were constricted (with arginine vasopressin or U46619) and relaxed (with bradykinin) before and after incubation with a phosphodiesterase-5 inhibitor, sildenafil citrate. We demonstrated increased myometrial small artery vasoconstriction and decreased endothelium-dependent vasodilatation in vessels from women whose pregnancies were complicated by FGR. Sildenafil citrate significantly reduced vasoconstriction and significantly improved relaxation of FGR small arteries. We conclude that sildenafil citrate improves endothelial function of myometrial vessels from women whose pregnancies are complicated by intrauterine growth restriction. Sildenafil citrate may offer a potential therapeutic strategy to improve uteroplacental blood flow in FGR pregnancies.

  3. The Methanolic Extract from Murraya koenigii L. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism

    Directory of Open Access Journals (Sweden)

    Nushrat Sharmin Ani

    2016-01-01

    Full Text Available Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant’s leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly (p<0.01 inhibited the pain thresholds induced by formalin and acetic acid in a dose-dependent manner. MEMK also significantly (p<0.01 suppressed glutamate-induced pain. Moreover, pretreatment with glibenclamide (an ATP-sensitive potassium channel blocker at 10 mg/kg significantly (p<0.05 reversed the MEMK-mediated antinociception. These revealed that MEMK might have the potential to interact with glutamatergic system and the ATP-sensitive potassium channels to exhibit its antinociceptive activities. Therefore, our results strongly support the antinociceptive effects of M. koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine.

  4. ATP Hydrolysis Induced Conformational Changes in the Vitamin B12 Transporter BtuCD Revealed by MD Simulations.

    Science.gov (United States)

    Pan, Chao; Weng, Jingwei; Wang, Wenning

    2016-01-01

    ATP binding cassette (ABC) transporters utilize the energy of ATP hydrolysis to uni-directionally transport substrates across cell membrane. ATP hydrolysis occurs at the nucleotide-binding domain (NBD) dimer interface of ABC transporters, whereas substrate translocation takes place at the translocation pathway between the transmembrane domains (TMDs), which is more than 30 angstroms away from the NBD dimer interface. This raises the question of how the hydrolysis energy released at NBDs is "transmitted" to trigger the conformational changes at TMDs. Using molecular dynamics (MD) simulations, we studied the post-hydrolysis state of the vitamin B12 importer BtuCD. Totally 3-μs MD trajectories demonstrate a predominantly asymmetric arrangement of the NBD dimer interface, with the ADP-bound site disrupted and the ATP-bound site preserved in most of the trajectories. TMDs response to ATP hydrolysis by separation of the L-loops and opening of the cytoplasmic gate II, indicating that hydrolysis of one ATP could facilitate substrate translocation by opening the cytoplasmic end of translocation pathway. It was also found that motions of the L-loops and the cytoplasmic gate II are coupled with each other through a contiguous interaction network involving a conserved Asn83 on the extended stretch preceding TM3 helix plus the cytoplasmic end of TM2/6/7 helix bundle. These findings entail a TMD-NBD communication mechanism for type II ABC importers.

  5. Low intracellular ATP levels exacerbate carcinogen-induced inflammatory stress response and inhibit in vitro tubulogenesis in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth Tahanian

    2011-01-01

    Full Text Available Elizabeth Tahanian, Sabrina Peiro, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de Recherche BioMED, Département de Chimie, Université du Québec à Montréal, Montréal, Québec, CanadaAbstract: Solid tumor development requires angiogenesis and is correlated to the expression of inflammatory markers through cellular metabolic and energetic adaptation. While high glycolysis rates enable the cancer cell compartment to generate adenosine triphosphate (ATP, very little is known about the impact of low intracellular ATP concentrations within the vascular endothelial cell compartment, which is responsible for tumor angiogenesis. Here, we investigated the effect of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis through intracellular ATP depletion, on human brain microvascular endothelial cell (HBMEC angiogenic properties. While preformed capillaries remained unaffected, we found that in vitro tubulogenesis was dose-dependently decreased by 2-DG and that this correlated with reduced intracellular ATP levels. Procarcinogenic signaling was induced with phorbol 12-myristate 13-acetate (PMA and found to trigger the proinflammatory marker cyclooxygenase-2 (COX-2 and endoplasmic reticulum (ER stress marker GRP78 expression, whose inductions were potentiated when PMA was combined with 2-DG treatment. Inversely, PMA-induced matrix-metalloproteinase-9 (MMP-9 gene expression and protein secretion were abrogated in the presence of 2-DG, and this can be partially explained by reduced nuclear factor-κB signaling. Collectively, we provide evidence for an intracellular ATP requirement in order for tubulogenesis to occur, and we link increases in ER stress to inflammation. A better understanding of the metabolic adaptations of the vascular endothelial cells that mediate tumor vascularization will help the development of new drugs and therapies.Keywords: endoplasmic reticulum stress, MMP-9, COX-2, 2-deoxy-D-glucose, endothelial

  6. Molecular dynamics simulation studies of GLUT4: substrate-free and substrate-induced dynamics and ATP-mediated glucose transport inhibition.

    Science.gov (United States)

    Mohan, Suma; Sheena, Aswathy; Poulose, Ninu; Anilkumar, Gopalakrishnapillai

    2010-12-03

    Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational

  7. Mechanisms of adenosine-induced renal vasodilatation in hypertensive patients.

    NARCIS (Netherlands)

    Wierema, T.K.; Houben, A.J.H.M.; Kroon, A.A.; Postma, C.T.; Koster, D.; Engelshoven, J.M. van; Smits, P.; Leeuw, P.W. de

    2005-01-01

    BACKGROUND: Adenosine is an endogenous nucleoside with potent vasodilatory capacities, released under ischaemic conditions in particular. Its mechanisms of action, however, remain elusive. OBJECTIVE: To evaluate the role of adenosine, using a non-selective purinergic receptor antagonist, and the

  8. Trainability of cold induced vasodilatation in fingers and toes

    NARCIS (Netherlands)

    Daanen, H.A.M.; Koedam, J.; Cheung, S.S.

    2012-01-01

    Subjects that repeatedly have to expose the extremities to cold may benefit from a high peripheral temperature to maintain dexterity and tissue integrity. Therefore, we investigated if repeated immersions of a hand and a foot in cold water resulted in increased skin temperatures. Nine male and seven

  9. Targeted Deletion of Adipocyte Abca1 (ATP-Binding Cassette Transporter A1) Impairs Diet-Induced Obesity.

    Science.gov (United States)

    Cuffe, Helen; Liu, Mingxia; Key, Chia-Chi C; Boudyguina, Elena; Sawyer, Janet K; Weckerle, Allison; Bashore, Alexander; Fried, Susan K; Chung, Soonkyu; Parks, John S

    2018-01-18

    Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity. Adiponectin Cre recombinase transgenic mice were crossed with Abca1flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis was similar between control and ASKO mice. Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1. © 2018 American Heart Association, Inc.

  10. Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin.

    Science.gov (United States)

    Zhang, Shiqiang; Chen, Shen; Li, Wen; Guo, Xiangpeng; Zhao, Ping; Xu, Jianyong; Chen, Yan; Pan, Qiong; Liu, Xiaorong; Zychlinski, Daniela; Lu, Hai; Tortorella, Micky D; Schambach, Axel; Wang, Yan; Pei, Duanqing; Esteban, Miguel A

    2011-08-15

    Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

  11. NFE2 Induces miR-423-5p to Promote Gluconeogenesis and Hyperglycemia by Repressing the Hepatic FAM3A-ATP-Akt Pathway.

    Science.gov (United States)

    Yang, Weili; Wang, Junpei; Chen, Zhenzhen; Chen, Ji; Meng, Yuhong; Chen, Liming; Chang, Yongsheng; Geng, Bin; Sun, Libo; Dou, Lin; Li, Jian; Guan, Youfei; Cui, Qinghua; Yang, Jichun

    2017-07-01

    Hepatic FAM3A expression is repressed under obese conditions, but the underlying mechanism remains unknown. This study determined the role and mechanism of miR-423-5p in hepatic glucose and lipid metabolism by repressing FAM3A expression. miR-423-5p expression was increased in the livers of obese diabetic mice and in patients with nonalcoholic fatty liver disease (NAFLD) with decreased FAM3A expression. miR-423-5p directly targeted FAM3A mRNA to repress its expression and the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic miR-423-5p inhibition suppressed gluconeogenesis and improved insulin resistance, hyperglycemia, and fatty liver in obese diabetic mice. In contrast, hepatic miR-423-5p overexpression promoted gluconeogenesis and hyperglycemia and increased lipid deposition in normal mice. miR-423-5p inhibition activated the FAM3A-ATP-Akt pathway and repressed gluconeogenic and lipogenic gene expression in diabetic mouse livers. The miR-423 precursor gene was further shown to be a target gene of NFE2, which induced miR-423-5p expression to repress the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic NFE2 overexpression upregulated miR-423-5p to repress the FAM3A-ATP-Akt pathway, promoting gluconeogenesis and lipid deposition and causing hyperglycemia in normal mice. In conclusion, under the obese condition, activation of the hepatic NFE2/miR-423-5p axis plays important roles in the progression of type 2 diabetes and NAFLD by repressing the FAM3A-ATP-Akt signaling pathway. © 2017 by the American Diabetes Association.

  12. Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane

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    Gaoyang Wang

    2018-01-01

    Full Text Available The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE. Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

  13. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    , we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca(2+) but, despite subsequent spikes in Ca(2+) concentration, no oscillations in oxidase activity could...

  14. ATP-binding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP.

    Science.gov (United States)

    Tsukamoto, K; Hirano, K; Tsujii, K; Ikegami, C; Zhongyan, Z; Nishida, Y; Ohama, T; Matsuura, F; Yamashita, S; Matsuzawa, Y

    2001-09-28

    Positional cloning approaches revealed that Tangier disease (TD), a genetic high density lipoprotein deficiency, is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) gene. However, the biological function of ABCA1 is still not fully investigated. Recently, we have reported that the cells from the patients with TD had abnormal actin cytoskeletons in association with decreased expression of Cdc42, a member of RhoGTPases family. In the present study, we have found that actin cytoskeletons were altered in HEK293 cells transfected with human ABCA1 (hABCA1) cDNA. Cells expressing hABCA1 were divided into the following two groups by the distinct morphology with altered actin cytoskeletons: one had increased formation of filopodia (designated as Type I) and the other had long protrusions (designated as Type II). Type I cells had morphology similar to that of cells transfected with dominant active form of Cdc42 (Cdc42-DA, V12Cdc42Hs-DA). Type II cells had morphology similar to that of cells transfected with neural Wiskott-Aldrich Syndrome Protein (N-WASP),one of the established downstream effector molecules of Cdc42. We have obtained the data showing a possible pathway of ABCA1/Cdc42/N-WASP by the following experiments. Introduction of mutant of Cdc42 (dominant negative form of Cdc42, N17Cdc42Hs-DN) and N-WASP (N-WASP lacking verprolin homology domain, N-WASPDeltaVPH), both of which are supposed to have potential to inhibit rearrangement of actin cytoskeletons, significantly inhibited the morphological changes induced by expression of hABCA1. Immunoprecipitation study with FLAG-tagged ABCA1 (hABCA1-FLAG) revealed that Cdc42 was coimmunoprecipitated with hABCA1-FLAG. In addition, we have demonstrated possible intracellular colocalization of these two molecules in the overexpressing cells by the confocal laser microscopy. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs. Copyright 2001

  15. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells.

    Science.gov (United States)

    Jiang, Kai; Wang, Wei; Jin, Xin; Wang, Zhaoyang; Ji, Zhiwei; Meng, Guanmin

    2015-06-01

    Silibinin, derived from the milk thistle plant (Silybum marianum), has anticancer and chemopreventive properties. Silibinin has been reported to inhibit the growth of various types of cancer cells. However, the mechanisms by which silibinin exerts an anticancer effect are poorly defined. The present study aimed to investigate whether silibinin-induced cell death might be attributed to autophagy and the underlying mechanisms in human MCF7 breast cancer cells. Our results showed that silibinin-induced cell death was greatly abrogated by two specific autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin-A1 (Baf-A1). In addition, silibinin triggered the conversion of light chain 3 (LC3)-I to LC3-II, promoted the upregulation of Atg12-Atg5 formation, increased Beclin-1 expression, and decreased the Bcl-2 level. Moreover, we noted elevated reactive oxygen species (ROS) generation, concomitant with the dissipation of mitochondrial transmembrane potential (ΔΨm) and a drastic decline in ATP levels following silibinin treatment, which were effectively prevented by the antioxidants, N-acetylcysteine and ascorbic acid. Silibinin stimulated the expression of Bcl-2 adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-death Bcl-2 family member, and silencing of BNIP3 greatly inhibited silibinin-induced cell death, decreased ROS production, and sustained ΔΨm and ATP levels. Taken together, these findings revealed that silibinin induced autophagic cell death through ROS-dependent mitochondrial dysfunction and ATP depletion involving BNIP3 in MCF7 cells.

  16. Molecular dynamics simulation studies of GLUT4: substrate-free and substrate-induced dynamics and ATP-mediated glucose transport inhibition.

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    Suma Mohan

    Full Text Available BACKGROUND: Glucose transporter 4 (GLUT4 is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states. Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. CONCLUSIONS/SIGNIFICANCE: In the absence of a crystal structure for any

  17. Pravastatin-induced improvement in coronary reactivity and circulating ATP and ADP levels in young adults with type 1 diabetes

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    Tuomas Oskari Kiviniemi

    2012-08-01

    Full Text Available Aims: Extracellular ATP and ADP regulate diverse inflammatory, prothrombotic and vasoactive responses in the vasculature. Statins have been shown to modulate their signaling pathways in vitro. We hypothesized that altered intravascular nucleotide turnover modulates vasodilation in patients with type 1 diabetes (T1DM, and this can be partly restored with pravastatin therapy. Methods: In this randomized double blind study, plasma ATP and ADP levels and echocardiography-derived coronary flow velocity response to cold pressor test (CPT were concurrently assessed in 42 normocholesterolemic patients with T1DM (age 30±6 years, LDL cholesterol 2.5±0.6 mmol/L before and after four-month treatment with pravastatin 40 mg/day or placebo (n=22 and n=20, respectively, and in 41 healthy control subjects. Results: Compared to controls, T1DM patients had significantly higher concentrations of ATP (p<0.01 and ADP (p<0.01 and these levels were partly restored after treatment with pravastatin (p=0.002 and p=0.007, respectively, but not after placebo (p=0.06 and p=0.14, respectively. Coronary flow velocity acceleration was significantly lower in T1DM patients compared to control subjects, and it increased from pre- to post-intervention in the pravastatin (p=0.02, but not in placebo group (p=0.15. Conclusions: Pravastatin treatment significantly reduces circulating ATP and ADP levels of T1DM patients, and concurrently improves coronary flow response to CPT. This study provides a novel insight in purinergic mechanisms involved in pleiotropic effects of pravastatin.

  18. Participation of the NO/cGMP/K{sup +}ATP pathway in the antinociception induced by Walker tumor bearing in rats

    Energy Technology Data Exchange (ETDEWEB)

    Barbosa, A.L.R.; Pinheiro, C.A.; Oliveira, G.J.; Torres, J.N.L.; Moraes, M.O.; Ribeiro, R.A.; Vale, M.L.; Souza, M.H.L.P. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil)

    2012-03-30

    Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K{sup +}ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K{sup +}ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E{sub 2}- (PGE{sub 2}, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE{sub 2}) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE{sub 2}) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE{sub 2}; 8 µg/paw) and the ATP-sensitive K{sup +} channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE{sub 2}; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

  19. Diltiazem Reduces Mortality and Breakdown of ATP in Red Blood Cell Induced by Isoproterenol in a Freely Moving Rat Model in Vivo

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    Pollen K.F. Yeung

    2014-09-01

    Full Text Available The benefit of calcium channel blockers for cardiovascular prevention against heart attack and stroke has not been firmly supported. We investigated the possible cardiovascular protective effect of diltiazem (DTZ against injury induced by isoproterenol using a freely moving rat model in vivo. Sprague Dawley rats were injected subcutaneously (sc with either 5 or 10 mg/kg of DTZ, or saline as control, twice daily for five doses. One hour after the last injection, a single dose of isoproterenol (30 mg/kg was injected sc to each rat. Blood samples were collected serially for 6 h for measurement of adenine nucleotides (ATP, ADP and AMP in red blood cell (RBC by a validated HPLC. The study has shown isoproterenol induced 50% mortality and also increased RBC concentrations of AMP from 0.04 ± 0.02 to 0.29 ± 0.21 mM at the end of the experiment (p < 0.05. Treatment with 10 mg/kg of DTZ reduced mortality from 50% to <20% and attenuated the increase of RBC concentrations of AMP from +0.25 ± 0.22 in the control rats to +0.072 ± 0.092 mM (p < 0.05. The study concluded that 10 mg/kg of DTZ reduced mortality and breakdown of ATP induced by isoproterenol in rats.

  20. Prevention of anti-microbial peptide LL-37-induced apoptosis and ATP release in the urinary bladder by a modified glycosaminoglycan.

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    Won Yong Lee

    Full Text Available Interstitial cystitis (IC, often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1 the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2 the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3 the physiological and molecular markers that correlate with the severity of the inflammation, and (4 the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.

  1. ATP Depletion Via Mitochondrial F1F0 Complex by Lethal Factor is an Early Event in B. Anthracis-Induced Sudden Cell Death

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    Mitchell W. Woodberry

    2009-08-01

    Full Text Available Bacillus anthracis’ primary virulence factor is a tripartite anthrax toxin consisting of edema factor (EF, lethal factor (LF and protective antigen (PA. In complex with PA, EF and LF are internalized via receptor-mediated endocytosis. EF is a calmodulin- dependent adenylate cyclase that induces tissue edema. LF is a zinc-metalloprotease that cleaves members of mitogen-activated protein kinase kinases. Lethal toxin (LT: PA plus LF-induced death of macrophages is primarily attributed to expression of the sensitive Nalp1b allele, inflammasome formation and activation of caspase-1, but early events that initiate these processes are unknown. Here we provide evidence that an early essential event in pyroptosis of alveolar macrophages is LF-mediated depletion of cellular ATP. The underlying mechanism involves interaction of LF with F1F0-complex gamma and beta subunits leading to increased ATPase activity in mitochondria. In support, mitochondrial DNA-depleted MH-S cells have decreased F1F0 ATPase activity due to the lack of F06 and F08 polypeptides and show increased resistance to LT. We conclude that ATP depletion is an important early event in LT-induced sudden cell death and its prevention increases survival of toxin-sensitive cells.

  2. Activation of ATP-sensitive potassium channel by iptakalim normalizes stress-induced HPA axis disorder and depressive behaviour by alleviating inflammation and oxidative stress in mouse hypothalamus.

    Science.gov (United States)

    Zhao, Xiao-Jie; Zhao, Zhan; Yang, Dan-Dan; Cao, Lu-Lu; Zhang, Ling; Ji, Juan; Gu, Jun; Huang, Ji-Ye; Sun, Xiu-Lan

    2017-04-01

    Stress-induced disturbance of the hypothalamic-pituitary-adrenal (HPA) axis is strongly implicated in incidence of mood disorders. A heightened neuroinflammatory response and oxidative stress play a fundamental role in the dysfunction of the HPA axis. We have previously demonstrated that iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could prevent oxidative injury and neuroinflammation against multiple stimuli-induced brain injury. The present study was to demonstrate the impacts of Ipt in stress-induced HPA axis disorder and depressive behavior. We employed 2 stress paradigms: 8 weeks of continuous restraint stress (chronic restraint stress, CRS) and 2h of restraint stress (acute restraint stress, ARS), to mimic both chronic stress and severe acute stress. Prolonged (4 weeks) and short-term (a single injection) Ipt treatment was administered 30min before each stress paradigm. We found that HPA axis was altered after stress, with different responses to CRS (lower ACTH and CORT, higher AVP, but normal CRH) and ARS (higher CRH, ACTH and CORT, but normal AVP). Both prolonged and short-term Ipt treatment normalized stress-induced HPA axis disorders and abnormal behaviors in mice. CRS and ARS up-regulated mRNA levels of inflammation-related molecules (TNFα, IL-1β, IL-6 and TLR4) and oxidative stress molecules (gp91phox, iNOS and Nrf2) in the mouse hypothalamus. Double immunofluorescence showed CRS and ARS increased microglia activation (CD11b and TNFα) and oxidative stress in neurons (NeuN and gp91phox), which were alleviated by Ipt. Therefore, the present study reveals that Ipt could prevent against stress-induced HPA axis disorders and depressive behavior by alleviating inflammation and oxidative stress in the hypothalamus. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Subclinical Doses of ATP-Sensitive Potassium Channel Modulators Prevent Alterations in Memory and Synaptic Plasticity Induced by Amyloid-β.

    Science.gov (United States)

    Salgado-Puga, Karla; Rodríguez-Colorado, Javier; Prado-Alcalá, Roberto A; Peña-Ortega, Fernando

    2017-01-01

    In addition to coupling cell metabolism and excitability, ATP-sensitive potassium channels (KATP) are involved in neural function and plasticity. Moreover, alterations in KATP activity and expression have been observed in Alzheimer's disease (AD) and during amyloid-β (Aβ)-induced pathology. Thus, we tested whether KATP modulators can influence Aβ-induced deleterious effects on memory, hippocampal network function, and plasticity. We found that treating animals with subclinical doses (those that did not change glycemia) of a KATP blocker (Tolbutamide) or a KATP opener (Diazoxide) differentially restrained Aβ-induced memory deficit, hippocampal network activity inhibition, and long-term synaptic plasticity unbalance (i.e., inhibition of LTP and promotion of LTD). We found that the protective effect of Tolbutamide against Aβ-induced memory deficit was strong and correlated with the reestablishment of synaptic plasticity balance, whereas Diazoxide treatment produced a mild protection against Aβ-induced memory deficit, which was not related to a complete reestablishment of synaptic plasticity balance. Interestingly, treatment with both KATP modulators renders the hippocampus resistant to Aβ-induced inhibition of hippocampal network activity. These findings indicate that KATP are involved in Aβ-induced pathology and they heighten the potential role of KATP modulation as a plausible therapeutic strategy against AD.

  4. ATP-sensitive K/sup +/ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone

    Energy Technology Data Exchange (ETDEWEB)

    de Weille, J.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1988-02-01

    The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide names from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and /sup 86/Rb/sup +/ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of APT-sensitive K/sup +/ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamine. Glibenclamide blocks the galanin-activated ATP-sensitive K/sup +/ channel. /sup 86/Rb/sup +/ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced /sup 86/Rb/sup +/ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K/sup +/ channel reported earlier. /sup 86/Rb/sup +/ efflux studies confirm the electrophysiological demonstration that galanin activates and ATP-dependent K/sup +/ channel.

  5. The Methanolic Extract fromMurraya koenigiiL. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+Channel as Antinociceptive Mechanism.

    Science.gov (United States)

    Sharmin Ani, Nushrat; Chakraborty, Sudip; Moniruzzaman, Md

    2016-01-01

    Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant's leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK) leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly ( p koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine.

  6. The Methanolic Extract from Murraya koenigii L. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism

    Science.gov (United States)

    Sharmin Ani, Nushrat; Chakraborty, Sudip

    2016-01-01

    Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant's leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK) leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly (p koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine. PMID:27812367

  7. Retrospectively gated MRI for in vivo assessment of endothelium-dependent vasodilatation and endothelial permeability in murine models of endothelial dysfunction.

    Science.gov (United States)

    Bar, Anna; Skórka, Tomasz; Jasiński, Krzysztof; Sternak, Magdalena; Bartel, Żaneta; Tyrankiewicz, Urszula; Chlopicki, Stefan

    2016-08-01

    Endothelial dysfunction is linked to impaired endothelial-dependent vasodilatation and permeability changes. Here, we quantify both of these phenomena associated with endothelial dysfunction by MRI in vivo in mice. Endothelial function was evaluated in the brachiocephalic artery (BCA) and left carotid artery (LCA) in ApoE/LDLR(-/-) and high-fat diet (HFD)-fed mice as compared with control mice (C57BL/6J). The 3D IntraGate® FLASH sequence was used for evaluation of changes in vessels' cross-sectional area (CSA) and volume following acetylcholine (Ach) administration. Evaluation of endothelial permeability after administration of contrast agent (Galbumin, BioPAL) was based on the variable flip angle method for the assessment of parameters based on the relaxation time (T1 ) value. In order to confirm the involvement of nitric oxide (NO) in response to Ach, L-NAME-treated mice were also analyzed. To confirm that endothelial permeability changes accompany the impairment of Ach-dependent vasodilatation, permeability changes were analyzed in isolated, perfused carotid artery. In C57BL/6J mice, Ach-induced vasodilatation led to an approximately 25% increase in CSA in both vessels, which was temporarily dissociated from the effect of Ach on heart rate. In ApoE/LDLR(-/-) or HFD-fed mice Ach induced a paradoxical vasoconstriction that amounted to approximately 30% and 50% decreases in CSA of BCA and LCA respectively. In ApoE/LDLR(-/-) and HFD-fed mice endothelial permeability in BCA was also increased (fall in T1 by about 25%). In L-NAME-treated mice Ach-induced vasodilatation in BCA was lost. In isolated, perfused artery from ApoE/LDLR(-/-) mice endothelial permeability was increased. MRI-based assessment of endothelium-dependent vasodilatation induced by Ach and endothelial permeability using a retrospectively self-gated 3D gradient-echo sequence (IntraGate® FLASH) enables the reliable detection of systemic endothelial dysfunction in mice and provides an important tool

  8. ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100.

    Science.gov (United States)

    White, I G; Voglmayr, J K

    1986-02-01

    It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in

  9. The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

    Science.gov (United States)

    Higashikuni, Yasutomi; Sainz, Julie; Nakamura, Kazuto; Takaoka, Minoru; Enomoto, Soichiro; Iwata, Hiroshi; Tanaka, Kimie; Sahara, Makoto; Hirata, Yasunobu; Nagai, Ryozo; Sata, Masataka

    2012-03-01

    ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

  10. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Camila Marques-da-Silva

    Full Text Available Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L. amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L. amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L. amazonensis escape mechanism is discussed.

  11. Differential Modulation of ATP-Induced P2X7-Associated Permeabilities to Cations and Anions of Macrophages by Infection with Leishmania amazonensis

    Science.gov (United States)

    Marques-da-Silva, Camila; Chaves, Mariana Martins; Rodrigues, Juliany Cola; Corte-Real, Suzana; Coutinho-Silva, Robson; Persechini, Pedro Muanis

    2011-01-01

    Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L.amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L.amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L.amazonensis escape mechanism is discussed. PMID:21966508

  12. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung

    2009-01-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... local concentration. Key words: sympathetic nerve activity, vasodilation, endothelium, skeletal muscle....

  13. Alendronate induces gastric damage by reducing nitric oxide synthase expression and NO/cGMP/K(ATP) signaling pathway.

    Science.gov (United States)

    Silva, Renan O; Lucetti, Larisse T; Wong, Deysi V T; Aragão, Karoline S; Junior, Eudmar M A; Soares, Pedro M G; Barbosa, André Luiz R; Ribeiro, Ronaldo A; Souza, Marcellus H L P; Medeiros, Jand-Venes R

    2014-08-31

    Chronic use of alendronate has been linked to gastrointestinal tract problems. Our objective was to evaluate the role of the NO/cGMP/KATP signaling pathway and nitric oxide synthase expression in alendronate-induced gastric damage. Rats were either treated with the NO donor, sodium nitroprusside (SNP; 1, 3, and 10 mg/kg), or the NO synthase (NOS) substrate, L-arginine (L-Arg; 50, 100, and 200 mg/kg). Some rats were pretreated with either ODQ (a guanylate cyclase inhibitor; 10 mg/kg) or glibenclamide (KATP channels blocker; 10 mg/kg). In other experiments, rats were pretreated with L-NAME (non-selective NOS inhibitor; 10 mg/kg), 1400 W (selective inducible NOS [iNOS] inhibitor; 10 mg/kg), or L-NIO (a selective endothelial NOS [eNOS] inhibitor; 30 mg/kg). After 1 h, the rats were treated with alendronate (30 mg/kg) by gavage for 4 days. SNP and L-Arg prevented alendronate-induced gastric damage in a dose-dependent manner. Alendronate reduced nitrite/nitrate levels, an effect that was reversed with SNP or L-Arg treatment. Pretreatment with ODQ or glibenclamide reversed the protective effects of SNP and L-Arg. L-NAME, 1400 W, or L-NIO aggravated the severity of alendronate-induced lesions. In addition, alendronate reduced the expression of iNOS and eNOS in the gastric mucosa. Gastric ulcerogenic responses induced by alendronate were mediated by a decrease in NO derived from both eNOS and iNOS. In addition, our findings support the hypothesis that activation of the NO/cGMP/KATP pathway is of primary importance for protection against alendronate-induced gastric damage. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Impact of catheter insertion using the radial approach on vasodilatation in humans.

    Science.gov (United States)

    Dawson, Ellen A; Rathore, Sudhir; Cable, N Timothy; Wright, D Jay; Morris, John L; Green, Daniel J

    2010-02-23

    The aim of this study was to determine the impact of catheter sheath insertion, a model of endothelium disruption in humans, on the conventional FMD (flow-mediated dilatation) response in vivo. Seventeen subjects undergoing transradial catheterization were recruited and assessed prior to, the day after, and 3-4 months postcatheterization. The catheter sheath's external diameter was 2.7 mm, and the average preprocedure internal radial artery diameter was 2.8 mm, indicating a high likelihood of endothelial denudation as a consequence of sheath placement. Radial artery flow-mediated and endothelium-derived NO (nitric oxide)-dependent function (FMD) was assessed within the region of sheath placement (sheath site) and also above the sheath (catheter site). GTN (glyceryl trinitrate) endothelium-independent NO-mediated function was also assessed distally. Measurements were made in both arms at all time points; the non-catheterized arm provided an internal control. Neither sheath (4.5+/-0.9%) nor catheter (4.4+/-0.9%) insertion abolished FMD, although both significantly decreased FMD from preintervention levels (9.0+/-0.8% sheath segment; 8.4+/-0.8% catheter segment; P<0.05). The impact of sheath and catheter placement on FMD was no longer evident after approximately 3 months recovery (8.0+/-1.5 and 8.1+/-1.7%, sheath and catheter, respectively). GTN responses also decreased from 14.8+/-1.7 to 7.9+/-1.0% (P<0.05) as a result of sheath placement, but values returned to baseline at approximately 3 months (13.0+/-1.8%). These results suggest that the presence of an intact, functional endothelial layer and consequent NO release may not be obligatory for some component of the FMD response. This raises the possibility of an endothelium-independent contribution to the flow-induced vasodilatation in humans.

  15. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Science.gov (United States)

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  16. Deficiency of ATP2C1, a golgi ion pump, induces secretory pathway defects in endoplasmic reticulum ( ER)-associated degradation and sensitivity to ER stress

    NARCIS (Netherlands)

    Ramos-Castaneda, J; Park, YN; Liu, M; Hauser, K; Rudolph, H; Shull, GE; Jonkman, MF; Mori, K; Ikeda, S; Ogawa, H; Arvan, P

    2005-01-01

    Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease ( Hailey-Hailey), which primarily affects

  17. Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+ signals and an IL-6 autocrine loop

    Science.gov (United States)

    Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique

    2014-01-01

    Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop. PMID:24518675

  18. ATP-Binding Cassette Transporter A1 Deficiency in Human Induced Pluripotent Stem Cell-Derived Hepatocytes Abrogates HDL Biogenesis and Enhances Triglyceride Secretion

    Directory of Open Access Journals (Sweden)

    Xin Bi

    2017-04-01

    Full Text Available Despite the recognized role of the ATP-binding Cassette Transporter A1 (ABCA1 in high-density lipoprotein (HDL metabolism, our understanding of ABCA1 deficiency in human hepatocytes is limited. To define the functional effects of human hepatocyte ABCA1 deficiency, we generated induced pluripotent stem cell (iPSC-derived hepatocyte-like cells (HLCs from Tangier disease (TD and matched control subjects. Control HLCs exhibited robust cholesterol efflux to apolipoprotein A-I (apoA-I and formed nascent HDL particles. ABCA1-deficient HLCs failed to mediate lipid efflux or nascent HDL formation, but had elevated triglyceride (TG secretion. Global transcriptome analysis revealed significantly increased ANGPTL3 expression in ABCA1-deficient HLCs. Angiopoietin-related protein 3 (ANGPTL3 was enriched in plasma of TD relative to control subjects. These results highlight the required role of ABCA1 in cholesterol efflux and nascent HDL formation by hepatocytes. Furthermore, our results suggest that hepatic ABCA1 deficiency results in increased hepatic TG and ANGPTL3 secretion, potentially underlying the elevated plasma TG levels in TD patients.

  19. Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for atp-citrate lyase in lipopolysaccharide-induced differentiation.

    Science.gov (United States)

    Dufort, Fay J; Gumina, Maria R; Ta, Nathan L; Tao, Yongzhen; Heyse, Shannon A; Scott, David A; Richardson, Adam D; Seyfried, Thomas N; Chiles, Thomas C

    2014-03-07

    Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.

  20. Caspase-independent apoptosis in Friend's erythroleukemia cells: role of mitochondrial ATP synthesis impairment in relocation of apoptosis-inducing factor and endonuclease G.

    Science.gov (United States)

    Comelli, Marina; Genero, Nadia; Mavelli, Irene

    2009-02-01

    Mitochondria have emerged as the central components of both caspase-dependent and independent apoptosis signalling pathways through release of different apoptogenic proteins. We previously documented that parental and differentiated Friend's erythroleukemia cells were induced to apoptosis by oligomycin and H(2)O(2) exposure, showing that the energy impairment occurring in both cases as a consequence of a severe mitochondrial F(0)F(1)ATPsynthase inactivation was a common early feature. Here we provide evidence for AIF and Endo G mitochondrio-nuclear relocation in both cases, as a component of caspase-independent apoptosis pathways. No detectable change in mitochondrial transmembrane potential and no variation in mitochondrial levels of Bcl-2 and Bax are observed. These results point to the osmotic rupture of the mitochondrial outer membrane as occurring in response to cell exposure to the two energy-impairing treatments under conditions preserving the mitochondrial inner membrane. A critical role of the mitochondrial F(0)F(1)ATP synthase inhibition in this process is also suggested.

  1. Caffeine dose-dependently induces thermogenesis but restores ATP in HepG2 cells in culture.

    Science.gov (United States)

    Riedel, Annett; Pignitter, Marc; Hochkogler, Christina M; Rohm, Barbara; Walker, Jessica; Bytof, Gerhard; Lantz, Ingo; Somoza, Veronika

    2012-09-01

    Caffeine has been hypothesised as a thermogenic agent that might help to maintain a healthy body weight. Since very little is known about its actions on cellular energy metabolism, we investigated the effect of caffeine on mitochondrial oxidative phosphorylation, cellular energy supply and thermogenesis in HepG2 cells, and studied its action on fatty acid uptake and lipid accumulation in 3T3-L1 adipocytes at concentrations ranging from 30-1500 μM. In HepG2 cells, caffeine induced a depolarisation of the inner mitochondrial membrane, a feature of mitochondrial thermogenesis, both directly and after 24 h incubation. Increased concentrations of uncoupling protein-2 (UCP-2) also indicated a thermogenic activity of caffeine. Energy generating pathways, such as mitochondrial respiration, fatty acid oxidation and anaerobic lactate production, were attenuated by caffeine treatment. Nevertheless, HepG2 cells demonstrated a higher energy charge potential after exposure to caffeine that might result from energy restoration through attenuation of energy consuming pathways, as typically found in hibernating animals. In 3T3-L1 cells, in contrast, caffeine increased fatty acid uptake, but did not affect lipid accumulation. We provide evidence that caffeine stimulates thermogenesis but concomitantly causes energy restoration that may compensate enhanced energy expenditure.

  2. Prevention of Diet-Induced Metabolic Dysregulation, Inflammation, and Atherosclerosis in Ldlr(-/-) Mice by Treatment With the ATP-Citrate Lyase Inhibitor Bempedoic Acid.

    Science.gov (United States)

    Samsoondar, Joshua P; Burke, Amy C; Sutherland, Brian G; Telford, Dawn E; Sawyez, Cynthia G; Edwards, Jane Y; Pinkosky, Stephen L; Newton, Roger S; Huff, Murray W

    2017-04-01

    Bempedoic acid (ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel low-density lipoprotein cholesterol-lowering compound. In animals, bempedoic acid targets the liver where it inhibits cholesterol and fatty acid synthesis through inhibition of ATP-citrate lyase and through activation of AMP-activated protein kinase. In this study, we tested the hypothesis that bempedoic acid would prevent diet-induced metabolic dysregulation, inflammation, and atherosclerosis. APPROACH AND RESULTS: Ldlr(-/-) mice were fed a high-fat, high-cholesterol diet (42% kcal fat, 0.2% cholesterol) supplemented with bempedoic acid at 0, 3, 10 and 30 mg/kg body weight/day. Treatment for 12 weeks dose-dependently attenuated diet-induced hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, fatty liver and obesity. Compared to high-fat, high-cholesterol alone, the addition of bempedoic acid decreased plasma triglyceride (up to 64%) and cholesterol (up to 50%) concentrations, and improved glucose tolerance. Adiposity was significantly reduced with treatment. In liver, bempedoic acid prevented cholesterol and triglyceride accumulation, which was associated with increased fatty acid oxidation and reduced fatty acid synthesis. Hepatic gene expression analysis revealed that treatment significantly increased expression of genes involved in fatty acid oxidation while suppressing inflammatory gene expression. In full-length aorta, bempedoic acid markedly suppressed cholesteryl ester accumulation, attenuated the expression of proinflammatory M1 genes and attenuated the iNos/Arg1 ratio. Treatment robustly attenuated atherosclerotic lesion development in the aortic sinus by 44%, with beneficial changes in morphology, characteristic of earlier-stage lesions. Bempedoic acid effectively prevents plasma and tissue lipid elevations and attenuates the onset of inflammation, leading to the prevention of atherosclerotic lesion development in a mouse model of metabolic

  3. Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

    Science.gov (United States)

    Denis, María Florencia Leal; Incicco, J. Jeremías; Espelt, María Victoria; Verstraeten, Sandra V.; Pignataro, Omar P.; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2014-01-01

    SUMMARY Background The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. Results Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General Significance Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. PMID:23742824

  4. Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yanni; Lai, Fangfang; Xu, Yang; Wu, Yexiang; Liu, Qi; Li, Ni; Wei, Yuzhen; Feng, Tingting; Zheng, Zhihui; Jiang, Wei; Yu, Liyan; Hong, Bin [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China); Si, Shuyi, E-mail: sisyimb@hotmail.com [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

  5. The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ramasri Sathanoori

    Full Text Available Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen

  6. Peripheral vasodilatation determines cardiac output in exercising humans

    DEFF Research Database (Denmark)

    Bada, A A; Svendsen, J H; Secher, N H

    2012-01-01

    In dogs, manipulation of heart rate has no effect on the exercise-induced increase in cardiac output. Whether these findings apply to humans remain uncertain, because of the large differences in cardiovascular anatomy and regulation. To investigate the role of heart rate and peripheral...

  7. Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

    Science.gov (United States)

    Ye, Xin-Chun; Hu, Jin-Xia; Li, Lei; Li, Qiang; Tang, Fu-Lei; Lin, Sen; Sun, Dong; Sun, Xiang-Dong; Cui, Gui-Yun; Mei, Lin; Xiong, Wen-Cheng

    2018-01-01

    Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. The brain-specific Lrp4 conditional knockout mice (Lrp4 GFAP-Cre ), astrocytic-specific Lrp4 conditional knockout mice (Lrp4 GFAP-creER ), and their control mice (Lrp4 f/f ) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A 2A R (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4 GFAP-Cre and Lrp4 GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P 2 X 7 R or adenosine-A 2A R signaling diminished Lrp4 GFAP-creER 's protective effect. The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A 2A R signaling. © 2017 American Heart

  8. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  9. Cold-acclimation-induced non-shivering thermogenesis in birds is associated with upregulation of avian UCP but not with innate uncoupling or altered ATP efficiency.

    Science.gov (United States)

    Teulier, Loïc; Rouanet, Jean-Louis; Letexier, Dominique; Romestaing, Caroline; Belouze, Maud; Rey, Benjamin; Duchamp, Claude; Roussel, Damien

    2010-07-15

    Despite their lack of brown adipose tissue, some bird species develop regulatory non-shivering thermogenesis (NST) of skeletal muscle origin in response to cold acclimation. Mechanisms involved in avian NST are still unclear but may involve reduced energetic coupling in skeletal muscle mitochondria through the expression of an avian homologue of mammalian uncoupling proteins. The aim of this work was to investigate whether the expression of avian uncoupling protein (avUCP) would correlate with the capacity for cold-induced muscle NST. Various levels of cold acclimation were obtained by rearing 1-week-old ducklings (Cairina moschata) for 4 weeks at three different ambient temperatures (25 degrees C, 11 degrees C or 4 degrees C). Muscle NST was measured by simultaneous recordings of metabolic rate and electromyographic activity (gastrocnemius muscle) at ambient temperatures (T(a)) ranging from 27 degrees C to -5 degrees C. The expression of avUCP gene and mitochondrial bioenergetics were also determined in gastrocnemius muscle. Results showed that muscle NST capacity depends on the T(a) at which ducklings were acclimated, i.e. the lower the rearing temperature, the higher the capacity for NST. This increased metabolic heat production occurred in parallel with an upregulation of avUCP, which was not associated with a change in mitochondrial membrane conductance. The intensity of mitochondrial oxidative phosphorylation also increased in proportion with the harshness of cold, while the efficiency of ATP generation was equally effective in all three acclimation temperatures. In the absence of mitochondrial uncoupling, these data indicate a clear link between avUCP expression and the capacity of ducklings to adjust their muscular aerobic activity to cold exposure.

  10. Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

    Science.gov (United States)

    Lee, Yung-Kuo; Lin, Tsung-Hsien; Chang, Chuan-Fa; Lo, Yu-Li

    2013-01-01

    Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells. PMID:24303084

  11. Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

    Science.gov (United States)

    Price, Alexander K; Fischer, David J; Martin, R Scott; Spence, Dana M

    2004-08-15

    The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

  12. A novel approach to regulate cell membrane permeability for ATP and NADH formation in Saccharomyces cerevisiae induced by air cold plasma

    Science.gov (United States)

    Dong, Xiaoyu; Liu, Tingting; Xiong, Yuqin

    2017-02-01

    Air cold plasma has been used as a novel method for enhancing microbial fermentation. The aim of this work was to explore the effect of plasma on membrane permeability and the formation of ATP and NADH in Saccharomyces cerevisiae, so as to provide valuable information for large-scale application of plasma in the fermentation industry. Suspensions of S. cerevisiae cells were exposed to air cold plasma for 0, 1, 2, 3, 4 and 5 min, and then subjected to various analyses prior to fermentation (0 h) and at the 9 and 21 h stages of fermentation. Compared with non-exposed cells, cells exposed to plasma for 1 min exhibited a marked increase in cytoplasmic free Ca2+ concentration as a result of the significant increase in membrane potential prior to fermentation. At the same time, the ATP level in the cell suspension decreased by about 40%, resulting in a reduction of about 60% in NADH prior to culturing. However, the levels of ATP and NADH in the culture at the 9 and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indicated that exposure of S. cerevisiae to air cold plasma could increase its cytoplasmic free Ca2+ concentration by improving the cell membrane potential, consequently leading to changes in ATP and NADH levels. Supported by National Natural Science Foundation of China (Nos. 21246012, 21306015 and 21476032).

  13. The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Cufi, Sílvia; Vellon, Luciano; Oliveras-Ferraros, Cristina; Menendez, Octavio J; Joven, Jorge; Lupu, Ruth; Menendez, Javier A

    2013-01-15

    Induced pluripotent stem (iPS) cells share some basic properties, such as self-renewal and pluripotency, with cancer cells, and they also appear to share several metabolic alterations that are commonly observed in human tumors. The cancer cells' glycolytic phenotype, first reported by Otto Warburg, is necessary for the optimal routing of somatic cells to pluripotency. However, how iPS cells establish a Warburg-like metabolic phenotype and whether the metabolic pathways that support the bioenergetics of iPS cells are produced by the same mechanisms that are selected during the tumorigenic process remain largely unexplored. We recently investigated whether the reprogramming-competent metabotype of iPS cells involves changes in the activation/expression status of the H(+)-ATPase, which is a core component of mitochondrial oxidative phosphorylation that is repressed at both the activity and protein levels in human carcinomas, and of the lipogenic switch, which refers to a marked overexpression and hyperactivity of the acetyl-CoA carboxylase (ACACA) and fatty acid synthase (FASN) lipogenic enzymes that has been observed in nearly all examined cancer types. A comparison of a starting population of mouse embryonic fibroblasts and their iPS cell progeny revealed that somatic cell reprogramming involves a significant increase in the expression of ATPase inhibitor factor 1 (IF1), accompanied by extremely low expression levels of the catalytic β-F1-ATPase subunit. The pharmacological inhibition of ACACA and FASN activities markedly decreases reprogramming efficiency, and ACACA and FASN expression are notably upregulated in iPS cells. Importantly, iPS cells exhibited a significant intracellular accumulation of neutral lipid bodies; however, these bodies may be a reflection of intense lysosomal/autophagocytic activity rather than bona fide lipid droplet formation in iPS cells, as they were largely unresponsive to pharmacological modulation of PPARgamma and FASN activities. The

  14. Diosgenin inhibits atherosclerosis via suppressing the MiR-19b-induced downregulation of ATP-binding cassette transporter A1.

    Science.gov (United States)

    Lv, Yun-cheng; Yang, Jing; Yao, Feng; Xie, Wei; Tang, Yan-yan; Ouyang, Xin-ping; He, Ping-ping; Tan, Yu-lin; Li, Liang; Zhang, Min; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Chao-ke

    2015-05-01

    Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms. Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn't affect liver X receptor α levels in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux, resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn. The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis

  15. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  16. Effects of ATP-induced leg vasodilation on VO2 peak and leg O2 extraction during maximal exercise in humans

    DEFF Research Database (Denmark)

    Calbet, J A L; Lundby, C; Sander, M

    2006-01-01

    During maximal whole body exercise VO2 peak is limited by O2 delivery. In turn, it is though that blood flow at near-maximal exercise must be restrained by the sympathetic nervous system to maintain mean arterial pressure. To determine whether enhancing vasodilation across the leg results in higher......) into the right femoral artery at a rate of 80 microg.kg body mass-1.min-1. During near-maximal exercise (92% of VO2 peak), the infusion of ATP increased leg vascular conductance (+43%, P...... O2 delivery and leg VO2 during near-maximal and maximal exercise in humans, seven men performed two maximal incremental exercise tests on the cycle ergometer. In random order, one test was performed with and one without (control exercise) infusion of ATP (8 mg in 1 ml of isotonic saline solution...

  17. Capsaicin-induced neurogenic inflammation in the skin in patients with symptoms induced by odorous chemicals

    DEFF Research Database (Denmark)

    Holst, Helle; Arendt-Nielsen, Lars; Mosbech, Holger

    2011-01-01

    Intradermal injection of capsaicin induces the axonal release of neuropeptides, vasodilatation and flare, e.g. neurogenic inflammation. The spatial profile of neurogenic inflammation in the skin has been studied in various experimental models. Polarization spectroscopy imaging introduced recently...

  18. Acute absolute vasodilatation is associated with a lower vascular wall stiffness in pulmonary arterial hypertension.

    Science.gov (United States)

    Grignola, Juan C; Domingo, Enric; Aguilar, Rio; Vázquez, Manuel; López-Messeguer, Manuel; Bravo, Carlos; Roman, Antonio

    2013-04-05

    Acute vasoreactivity testing (VT) is considered mandatory in the diagnostic work-up of patients with pulmonary arterial hypertension (PAH). We studied the relation between the acute absolute arterial vasodilatation and the severity of vascular remodeling estimated by intravascular ultrasound (IVUS) in patients with idiopathic PAH. Simultaneous right heart catheterization and IVUS of the pulmonary artery (PA) were performed both in basal conditions and during short-term intravenous epoprostenol infusion in nineteen idiopathic PAH patients. Pulmonary vascular resistance (PVRi) and capacitance indexes (stroke volume/pulse pressure, Cp), were calculated. Local pulsatility was estimated by IVUS (IVUSp) (systolic-diastolic lumen area/diastolic lumen area×100; sA-dA/dA) and PA stiffness was assessed by the elastic modulus (E: pulse pressure/IVUSp). Epoprostenol infusion (11±2ng/kg/min) determined a real vasodilatation (increment of dA>10%) in six patients. This vasodilation group presented on average significantly higher cardiac index, stroke volume index and Cp, and lower PVRi and IVUSp (Prights reserved.

  19. Noradrenaline and neuropeptide Y contribute to initial, but not sustained, vasodilatation in response to local skin warming in humans.

    Science.gov (United States)

    Hodges, Gary J; Sparks, Paul A

    2014-02-01

    What is the central question of this study? Previous work has produced the counterintuitive finding that the vasoconstrictor neurotransmitters noradrenaline and neuropeptide Y are involved in vasodilatation. We aimed to discover whether sympathetic neurotransmitters are required for the sustained vasodilatation in response to local skin warming, as has been previously suggested, and to determine whether noradrenaline and neuropeptide Y are 'mediating' the sustained vasodilator response directly or acting to 'prime' (or kick-start) it. What is the main finding and its importance? We have found that noradrenaline and neuropeptide Y are required at the initiation of vasodilatation in response to local skin warming, if a complete vasodilator response is to be achieved; however, they are not required once vasodilatation has begun. In a three-part study, we examined whether noradrenaline, neuropeptide Y (NPY) and endothelial nitric oxide synthase (eNOS) were involved in the sustained vasodilatation in response to local skin warming. Forearm skin sites were instrumented with intradermal microdialysis fibres, local skin heaters and laser-Doppler flow probes. Local skin temperature (T(loc)) was increased from 34 to 42°C at a rate of 0.5°C (10 s)(-1). Laser-Doppler flow was expressed as cutaneous vascular conductance (CVC; laser-Doppler flow/mean arterial pressure). In part 1, three skin sites were prepared; two were treated with the study vehicle (lactated Ringer solution), while the third site was treated with yohimbine and propranolol to antagonize α- and β-receptors, and 10 min of baseline data were record at a T(loc) of 34°C. Receptor antagonism was confirmed via infusion of clonidine. The T(loc) was increased to 42°C at all sites. Once CVC had stabilized, site 2 was treated with yohimbine and propranolol to examine the effect of adrenergic receptor blockade on sustained vasodilatation of the skin. Receptor antagonism was again confirmed via infusion of clonidine

  20. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  1. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  2. Fasting induces anti-Warburg effect that increases respiration but reduces ATP-synthesis to promote apoptosis in colon cancer models.

    Science.gov (United States)

    Bianchi, Giovanna; Martella, Roberto; Ravera, Silvia; Marini, Cecilia; Capitanio, Selene; Orengo, Annamaria; Emionite, Laura; Lavarello, Chiara; Amaro, Adriana; Petretto, Andrea; Pfeffer, Ulrich; Sambuceti, Gianmario; Pistoia, Vito; Raffaghello, Lizzia; Longo, Valter D

    2015-05-20

    Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called "Warburg effect" whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O(2) consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex II-dependent O(2) consumption, elevated oxidative stress and apoptosis .These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.

  3. Transradial catheterization may decrease the radial artery luminal diameter and impair the vasodilatation response in the access site at late term: an observational study.

    Science.gov (United States)

    Buturak, Ali; Tekturk, Burak Murat; Degirmencioglu, Aleks; Ulus, Sila; Surgit, Ozgur; Ariturk, Cem; Zencirci, Ertugrul; Gorgulu, Sevket

    2016-04-01

    The present study aimed to evaluate the late-term changes in radial artery luminal diameter (RAD) and vasodilatation response following transradial catheterization (TRC). TRC-inducing trauma to radial artery intima may trigger chronic phase vascular changes and lead to anatomical and functional impairment. There is controversial data whether the impairment persists or repairs later. Fifty-six consecutive patients undergoing TRC were enrolled prospectively. Baseline RAD, flow-mediated dilatation (FMD) and nitroglycerin-mediated dilatation (NMD) of the radial artery at the access site were measured before TRC by high-resolution ultrasound. Six months later; RAD, FMD and NMD were measured again at the same access site. RAD at the sixth month was reduced compared with pre-procedural measurements (2.85 ± 0.44 versus 2.74 ± 0.42 mm, p = 0.0001).The average FMD decreased to 5.66 ± 5.87 %, which was significantly lower than the observed pre-procedural FMD (9.45 ± 5.01 %) 6 months after TRC (p = 0.0001). Likewise, the average NMD at the sixth month was reduced compared with pre-procedural NMD (9.52 ± 6.77 versus 6.64 ± 6.51 %, p = 0.018). Logistic regression analysis indicated that pre-procedural radial artery diameter to sheath size ratio was the independent predictor of NMD reduction (95 % confidence interval, β = -9.74, p = 0.024). TRC may lead to a significant luminal diameter reduction and impairment of vasodilatation response in the radial artery at late term.

  4. Novel approaches to improving endothelium-dependent nitric oxide-mediated vasodilatation

    DEFF Research Database (Denmark)

    Simonsen, Ulf; Rodriguez-Rodriguez, Rosalia; Dalsgaard, Thomas

    2009-01-01

    Endothelial dysfunction, which is defined by decreased endothelium-dependent vasodilatation, is associated with an increased number of cardiovascular events. Nitric oxide (NO) bioavailability is reduced by altered endothelial signal transduction or increased formation of radical oxygen species...... reacting with NO. Endothelial dysfunction is therapeutically reversible and physical exercise, calcium channel blockers, angiotensin converting enzyme inhibitors, and angiotensin receptor antagonists improve flow-evoked endothelium-dependent vasodilation in patients with hypertension and diabetes. We have...... investigated three different approaches, with the aim of correcting endothelial dysfunction in cardiovascular disease. Thus, (1) we evaluated the effect of a cell permeable superoxide dismutase mimetic, tempol, on endothelial dysfunction in small arteries exposed to high pressure, (2) investigated...

  5. Vasodilatation of afferent arterioles and paradoxical increase of renal vascular resistance by furosemide in mice

    DEFF Research Database (Denmark)

    Oppermann, Mona; Hansen, Pernille B; Castrop, Hayo

    2007-01-01

    Loop diuretics like furosemide have been shown to cause renal vasodilatation in dogs and humans, an effect thought to result from both a direct vascular dilator effect and from inhibition of tubuloglomerular feedback. In isolated perfused afferent arterioles preconstricted with angiotensin II or N......(G)-nitro-L-arginine methyl ester, furosemide caused a dose-dependent increase of vascular diameter, but it was without effect in vessels from NKCC1-/- mice suggesting that inhibition of NKCC1 mediates dilatation in afferent arterioles. In the intact kidney, however, furosemide (2 mg/kg iv) caused a 50.5 +/- 3% reduction...... of total renal blood flow (RBF) and a 27% reduction of superficial blood flow (SBF) accompanied by a marked and immediate increase of tubular pressure and volume. At 10 mg/kg, furosemide reduced RBF by 60.4 +/- 2%. Similarly, NKCC1-/- mice responded to furosemide with a 45.4% decrease of RBF and a 29...

  6. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    Science.gov (United States)

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  7. Carbon monoxide inhalation induces headache in a human headache model

    DEFF Research Database (Denmark)

    Arngrim, Nanna; Schytz, Henrik Winther; Britze, Josefine

    2017-01-01

    Introduction Carbon monoxide (CO) is an endogenously produced signalling molecule that has a role in nociceptive processing and cerebral vasodilatation. We hypothesized that inhalation of CO would induce headache and vasodilation of cephalic and extracephalic arteries. Methods In a randomized...

  8. Heat-provoked skin vasodilatation in innervated and denervated trunk dermatomes in human spinal cord injury.

    Science.gov (United States)

    Nicotra, A; Asahina, M; Young, T M; Mathias, C J

    2006-04-01

    Cross-sectional, observational, controlled study. High spinal cord injury (SCI) results in disruption of sympathetic vasomotor control. Vasodilatation as a response to local heating is a biphasic mechanism: the first phase (neurogenic) is mediated by the axon-reflex and is modulated by activity of sympathetic nerves. Our objective was to determine whether the response to heat provocation in trunk dermatomes may provide a measure of vasomotor sympathetic function in SCI. National Spinal Injuries Centre, Stoke Mandeville Hospital, Buckinghamshire, UK; Autonomic Unit, The National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; Neurovascular Medicine Unit, Imperial College London at St Mary's Hospital, UK. A total of 30 subjects were studied; 18 had chronic complete SCI (level C6-T11) and 12 were healthy controls. Recordings of skin blood flow (SkBF) were obtained with thermostatic laser Doppler probes placed in the upper trunk (at C4) and lower trunk (T10 or T12) dermatomes. SkBF at baseline (SkBF(bas)) and SkBF at the first peak of vasodilatation (SkBF(max)) showed no significant differences between SCI and controls either in upper or lower trunk dermatomes. However, the ratio of SkBF(max)/SkBF(bas) was significantly different in lower trunk dermatomes in SCI at C6-T5 level (7.5+/-3.5 PU) compared to SCI at T6-T11 level (3.5+/-1.5 PU) (P < 0.01). Measurement of SkBF in response to local heating may provide a safe, noninvasive method to assess integrity of sympathetic spinal pathways to the local vasculature. This may aid the classification of the SCI lesions, as the autonomic component currently is not included in the accepted American Spinal Injury Association scoring.

  9. Association of the α2δ1 Subunit with Cav3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

    Science.gov (United States)

    Thompson, William R.; Majid, Amber S.; Czymmek, Kirk J.; Ruff, Albert L.; García, Jesús; Duncan, Randall L.; Farach-Carson, Mary C.

    2015-01-01

    Voltage sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α1 pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs, but a relatively small amount of L-type α1 subunits. Auxiliary VSCC subunits have several functions including modulating gating kinetics, trafficking of the channel and phosphorylation events. The influence of the α2δ auxiliary subunit on T-type VSCCs and the physiological consequences of that association are incompletely understood and have yet to be investigated in bone. In this study, we postulated that the auxiliary α2δ subunit of the VSCC complex modulates mechanically-regulated ATP release in osteocytes via its association with the T-type, Cav3.2 (α1H) subunit. We demonstrated by RT-PCR, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type, Cav3.2 (α1H) subunit more abundantly than the L-type, Cav1.2 (α1C). We also demonstrated that the α2δ1 subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Cav3.2 (α1H) subunit in MLO-Y4 cells. Interestingly, siRNA mediated knockdown of α2δ1 completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α2δ1 subunit and resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α2δ1 knockdown showed decreased membrane localization of Cav3.2 (α1H) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Cav3.2 (α1H) at the cell membrane. PMID:21638318

  10. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  11. Modulation of postjunctional α-adrenergic vasoconstriction during exercise and exogenous ATP infusions in ageing humans

    Science.gov (United States)

    Kirby, Brett S; Crecelius, Anne R; Voyles, Wyatt F; Dinenno, Frank A

    2011-01-01

    Abstract The ability to modulate sympathetic α-adrenergic vasoconstriction in contracting muscle is impaired with age. In young adults, adenosine triphosphate (ATP) has been shown to blunt sympathetic vasoconstrictor responsiveness similar to exercise. Therefore, we tested the hypothesis that modulation of postjunctional α-adrenergic vasoconstriction to exogenous ATP is impaired in ageing humans. We measured forearm blood flow (FBF; Doppler ultrasound) and calculated vascular conductance (FVC) to intra-arterial infusions of phenylephrine (α1-agonist) and dexmedetomidine (α2-agonist) during rhythmic handgrip exercise (15% MVC), a control non-exercise vasodilator condition (adenosine), and ATP infusion in seven older (64 ± 3 years) and seven young (22 ± 1 years) healthy adults. Forearm hyperaemia was matched across all vasodilatating conditions. During adenosine, forearm vasoconstrictor responses to direct α1-stimulation were lower in older compared with young adults (ΔFVC =−25 ± 3%vs.−41 ± 5%; P different (−35 ± 6%vs.−44 ± 8%; NS). During exercise, α1-mediated vasoconstriction was significantly blunted compared with adenosine in both young (−9 ± 2%vs.−41 ± 5%) and older adults (−15 ± 2%vs.−25 ± 3%); however, the magnitude of sympatholysis was reduced in older adults (32 ± 13 vs. 74 ± 8%; P exercise was significantly blunted in both young (−15 ± 4%vs.−44 ± 8%) and older adults (−26 ± 3%vs.−35 ± 6%), however the magnitude of sympatholysis was reduced in older adults (19 ± 8%vs. 60 ± 10%; P age groups (∼85–90%). Our findings indicate that the ability to modulate postjunctional α-adrenergic vasoconstriction during exercise is impaired with age, whereas the sympatholytic effect of exogenous ATP is preserved. Thus, if impairments in vascular control during exercise in older adults involve vasoactive ATP, we speculate that circulating ATP is reduced with advancing age. PMID:21486772

  12. Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes.

    Science.gov (United States)

    He, Z; Leong, D J; Zhuo, Z; Majeska, R J; Cardoso, L; Spray, D C; Goldring, M B; Cobelli, N J; Sun, H B

    2016-05-01

    To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the

  13. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

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    M Florencia Leal Denis

    Full Text Available The peptide mastoparan 7 (MST7 triggered in human erythrocytes (rbcs the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe, interacting with P (purinergic receptors, can affect cell volume (Vr, we explored the dynamic regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

  14. Central venous hypoxemia is a determinant of human atrial ATP-sensitive potassium channel expression: evidence for a novel hypoxia-inducible factor 1alpha-Forkhead box class O signaling pathway.

    Science.gov (United States)

    Raeis, Véronique; Philip-Couderc, Pierre; Roatti, Angela; Habre, Walid; Sierra, Jorge; Kalangos, Afksendyios; Beghetti, Maurice; Baertschi, Alex J

    2010-05-01

    ATP-sensitive potassium channels couple cell excitability to energy metabolism, thereby providing life-saving protection of stressed cardiomyocytes. The signaling for ATP-sensitive potassium channel expression is still unknown. We tested involvement of biochemical and biophysical parameters and potential transcription factors Forkhead box (FOX) and hypoxia-inducible factor (HIF-1alpha). Right atrial tissues were obtained during surgery from 28 children with heart disease. Expression of K(+)-inward-rectifier subunits Kir6.1/Kir6.2; sulfonyl urea receptors (SURs) SUR1A/B and SUR2A/B; and FOX class O (FOXO) 1, FOXO3, FOXF2, and HIF-1alpha were related to 31 parameters, including personal data, blood chemistry, and echocardiography. Venous hypoxemia (but not other ischemia indicators, such as venous hypercapnia or low glucose) predicts increased Kir6.1 (Phypoxemia (P<0.003). Electrophoretic mobility-shift assays suggest causal links among hypoxia, HIF-1alpha, FOXO1, and Kir6.1. To mimic mild ischemia encountered in some patients, cultured rat atrial myocytes were tested in hypoxia, hypercapnia, or low glucose, with normal conditions serving as the control. Mild hypoxia (24-hour) increases expression of HIF-1alpha, FOXO1, and SUR2A/B/Kir6.1 in culture (P<0.01), whereas hypercapnia and low glucose have no or opposite effects. Gene knockdown of HIF-1alpha or FOXO1 by small-interfering RNAs abolishes hypoxia-induced expression of FOXO1 and SUR2A/B/Kir6.1. These results suggest that low tissue oxygen determines increased expression of the atrial SUR2A/B/Kir6.1 gene via activation of HIF-1alpha-FOXO1. Because increased SUR2A/B/Kir6.1 has known survival benefits, this pathway offers novel therapeutic targets for children with heart disease.

  15. Vasodilatation induced by croton schiedeanus schlecht associated with metabolic way of guanylate cyclase

    OpenAIRE

    Chaves Torres, Marlén; Puebla Ibáñez, Pilar; Guerrero Pabón, Mario

    2012-01-01

    The EtOH extract of Croton shiedeanus Schlecht, medicinal species used by the popular tradition in Colombia for hypertension, relaxes vascular rings of Wistar rat aorta stimulated with phenylephrine (10-6 M, alpha adrenergic receptor agonist), in a dose-depending manner, with an EC50 of 1.3 × 10-5 g/mL. This effect is superior to that exercised over rings stimulated with KCl (6,0 × 10-2 M, voltage-dependent calcium channel activator) and is reduced in vascular rings without endothelium. The v...

  16. Lack of Correlation Between Vasodilatation and Pharmacologically Induced Immediate Headache in Healthy Subjects

    DEFF Research Database (Denmark)

    Ashina, Messoud; Tfelt-Hansen, Peer; Dalgaard, Peter

    2011-01-01

    these experiments were vasodilators we examined a possible correlation between headache scores and increases in arterial diameter. Methods: We identified nine studies and retrieved raw data in 89 healthy subjects (46 females, 43 males), mean age 27 years (range 18–59 years). The following variables were collected......: maximal median headache intensity scores on a verbal rating scale (VRS) during immediate headache (0–120 minutes); the mean velocity of blood flow in the middle cerebral artery (VmeanMCA); and the diameter of the frontal branch of the superficial temporal artery (STA) during the maximal median headache...... intensity. Results: The scatter plots show no relationship between maximal headache score and the relative changes in VmeanMCA and diameter of the STA. The main analyses of covariance showed a significant effect only of heart rate on headache (p = .014). The interaction tests were insignificant for all...

  17. Nanomolar ambient ATP decelerates P2X3 receptor kinetics.

    Science.gov (United States)

    Grote, Alexander; Hans, Michael; Boldogkoi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

    2008-12-01

    Homomeric P2X receptors differ in their electrophysiological and pharmacological profiles. The rapidly activating and desensitizing P2X3 receptors are known for their involvement in pain signalling pathways. Modulatory effects on P2X3 receptors have been reported for low concentrations of ATP ([ATP]). This includes both, enhancement and reduction of receptor currents. The first has been reported to be mediated by activation of ectoprotein kinases and high affinity desensitization (HAD), respectively. Both processes influence receptor current amplitudes. Here we describe a new phenomenon, the modulatory influence of ambient low [ATP] on P2X3 receptor kinetics. First, we studied in HEK cells whether persistent ATP affects current decay. To this end, P2X3 receptor mediated currents, elicited by pressure application of saturating [ATP], were analyzed after pre-application of low [ATP]. Second, UV-flash photolysis of ATP was employed to investigate whether submicromolar [ATP] affects receptor activation. Finally we confirmed the action of nanomolar [ATP] on native P2X3 receptors of neurons freshly isolated from rat dorsal root ganglia. We found that persistent low [ATP] caused pronounced deceleration of receptor current activation and decay. This priming effect indicates a mechanism different from HAD. It could be explained by a pre-opening receptor isomerization, induced by the occupation of a high affinity binding site already at the resting state. The observed modulation of the receptor kinetics could be considered as a physiological fine tuning mechanism of the nociceptive system, driven by the actual ambient agonist concentration.

  18. Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

    Directory of Open Access Journals (Sweden)

    Ip Virginia

    2010-09-01

    Full Text Available Abstract Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H. High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non

  19. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

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    Romina Baaske

    2016-12-01

    Full Text Available Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla. This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L, which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.

  20. P2Y Receptor Modulation of ATP Release in the Urothelium

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    Kylie J. Mansfield

    2014-01-01

    Full Text Available The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells. Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8 µM. Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM. Selective P2 receptor agonists, αβ-methylene-ATP, ATP-γ-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100 µM, P = 0.002. RT4 urothelial cells break down nucleotides (100 µM including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.

  1. Rebalancing β-Amyloid-Induced Decrease of ATP Level by Amorphous Nano/Micro Polyphosphate: Suppression of the Neurotoxic Effect of Amyloid β-Protein Fragment 25-35

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    Werner E. G. Müller

    2017-10-01

    Full Text Available Morbus Alzheimer neuropathology is characterized by an impaired energy homeostasis of brain tissue. We present an approach towards a potential therapy of Alzheimer disease based on the high-energy polymer inorganic polyphosphate (polyP, which physiologically occurs both in the extracellular and in the intracellular space. Rat pheochromocytoma (PC 12 cells, as well as rat primary cortical neurons were exposed to the Alzheimer peptide Aβ25-35. They were incubated in vitro with polyphosphate (polyP; ortho-phosphate was used as a control. The polymer remained as Na+ salt; or complexed in a stoichiometric ratio to Ca2+ (Na-polyP[Ca2+]; or was processed as amorphous Ca-polyP microparticles (Ca-polyP-MP. Ortho-phosphate was fabricated as crystalline Ca-phosphate nanoparticles (Ca-phosphate-NP. We show that the pre-incubation of PC12 cells and primary cortical neurons with polyP protects the cells against the neurotoxic effect of the Alzheimer peptide Aβ25-35. The strongest effect was observed with amorphous polyP microparticles (Ca-polyP-MP. The effect of the soluble sodium salt; Na-polyP (Na-polyP[Ca2+] was lower; while crystalline orthophosphate nanoparticles (Ca-phosphate-NP were ineffective. Ca-polyP-MP microparticles and Na-polyP[Ca2+] were found to markedly enhance the intracellular ATP level. Pre-incubation of Aβ25-35 during aggregate formation, with the polyP preparation before exposure of the cells, had a small effect on neurotoxicity. We conclude that recovery of the compromised energy status in neuronal cells by administration of nontoxic biodegradable Ca-salts of polyP reverse the β-amyloid-induced decrease of adenosine triphosphate (ATP level. This study contributes to a new routes for a potential therapeutic intervention in Alzheimer’s disease pathophysiology.

  2. ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

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    Francesco Drago

    2017-12-01

    Full Text Available Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs, which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012. However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

  3. An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts

    DEFF Research Database (Denmark)

    Pateraki, Irini; Renato, Marta; Azcõn-Bieto, Joaquín

    2013-01-01

    Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active...... synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes...... the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize...

  4. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    Science.gov (United States)

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

  5. Non-linear relationship between hyperpolarisation and relaxation enables long distance propagation of vasodilatation.

    Science.gov (United States)

    Wölfle, Stephanie E; Chaston, Daniel J; Goto, Kenichi; Sandow, Shaun L; Edwards, Frank R; Hill, Caryl E

    2011-05-15

    Blood flow is adjusted to tissue demand through rapidly ascending vasodilatations resulting from conduction of hyperpolarisation through vascular gap junctions. We investigated how these dilatations can spread without attenuation if mediated by an electrical signal. Cremaster muscle arterioles were studied in vivo by simultaneously measuring membrane potential and vessel diameter. Focal application of acetylcholine elicited hyperpolarisations which decayed passively with distance from the local site,while dilatation spread upstream without attenuation. Analysis of simultaneous recordings at the local site revealed that hyperpolarisation and dilatation were only linearly related over a restricted voltage range to a threshold potential, beyond which dilatation was maximal. Experimental data could be simulated in a computational model with electrotonic decay of hyperpolarisation but imposition of this threshold. The model was tested by reducing the amplitude of the local hyperpolarisation which led to entry into the linear range closer to the local site and decay of dilatation. Serial section electron microscopy and light dye treatment confirmed that the spread of dilatation occurred through the endothelium and that the two cell layers were tightly coupled. Generality of the mechanism was demonstrated by applying the model to the attenuated propagation of dilatation found in larger arteries.We conclude that long distance spread of locally initiated dilatations is not due to a regenerative electrical phenomenon, but rather a restricted linear relationship between voltage and vessel tone, which minimises the impact of electrotonic decay of voltage. Disease-related alterations in endothelial coupling or ion channel expression could therefore decrease the ability to adjust blood flow to meet metabolic demand.

  6. Blunted muscle vasodilatation during chemoreceptor stimulation in patients with heart failure.

    Science.gov (United States)

    Di Vanna, Andrea; Braga, Ana Maria F W; Laterza, Mateus C; Ueno, Linda M; Rondon, Maria Urbana P B; Barretto, Antonio C P; Middlekauff, Holly R; Negrão, Carlos E

    2007-07-01

    Chemoreflex control of sympathetic nerve activity is exaggerated in heart failure (HF) patients. However, the vascular implications of the augmented sympathetic activity during chemoreceptor activation in patients with HF are unknown. We tested the hypothesis that the muscle blood flow responses during peripheral and central chemoreflex stimulation would be blunted in patients with HF. Sixteen patients with HF (49 +/- 3 years old, Functional Class II-III, New York Heart Association) and 11 age-paired normal controls were studied. The peripheral chemoreflex control was evaluated by inhalation of 10% O(2) and 90% N(2) for 3 min. The central chemoreflex control was evaluated by inhalation of 7% CO(2) and 93% O(2) for 3 min. Muscle sympathetic nerve activity (MSNA) was directly evaluated by microneurography. Forearm blood flow was evaluated by venous occlusion plethysmography. Baseline MSNA were significantly greater in HF patients (33 +/- 3 vs. 20 +/- 2 bursts/min, P = 0.001). Forearm vascular conductance (FVC) was not different between the groups. During hypoxia, the increase in MSNA was significantly greater in HF patients than in normal controls (9.0 +/- 1.6 vs. 0.8 +/- 2.0 bursts/min, P = 0.001). The increase in FVC was significantly lower in HF patients (0.00 +/- 0.10 vs. 0.76 +/- 0.25 units, P = 0.001). During hypercapnia, MSNA responses were significantly greater in HF patients than in normal controls (13.9 +/- 3.2 vs. 2.1 +/- 1.9 bursts/min, P = 0.001). FVC responses were significantly lower in HF patients (-0.29 +/- 0.10 vs. 0.37 +/- 0.18 units, P = 0.001). In conclusion, muscle vasodilatation during peripheral and central chemoreceptor stimulation is blunted in HF patients. This vascular response seems to be explained, at least in part, by the exaggerated MSNA responses during hypoxia and hypercapnia.

  7. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism.

    Science.gov (United States)

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2015-08-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Role of the NO/K ATP pathway in the protective effect of a sulfated-polysaccharide fraction from the algae Hypnea musciformis against ethanol-induced gastric damage in mice

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    Samara R. B. Damasceno

    2013-02-01

    Full Text Available Seaweeds are the most abundant source of polysaccharides such as alginates and agar, as well as carrageenans. This study aimed to investigate the gastroprotective activity and the mechanism underlying this activity of a sulfated-polysaccharide fraction extracted from the algae Hypnea musciformis (Wulfen J.V. Lamour. (Gigartinales-Rhodophyta. Mice were treated with sulfated-polysaccharide fraction (3, 10, 30, and 90 mg/kg, p.o. and, after 30 min, they were administered 50% ethanol (0.5 mL/25 g, p.o.. After 1 h, gastric damage was measured using a planimeter. In addition, samples of the stomach tissue were obtained for histopathological examination and for assays to determine the glutathione and malondialdehyde levels. Other groups of mice were pretreated with N G-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.p., aminoguanidine (100 mg/kg, i.p., or glibenclamide (10 mg/kg, i.p.. After 30 min to the aminoguanidine group and 1 h to the other groups, sulfated-polysaccharide fraction (30 mg/kg, p.o. was administered and gastric damage was induced as described above. Sulfated-polysaccharide fraction prevented ethanol-induced gastric injury in a dose-dependent manner. However, treatment with L-NAME or glibenclamide reversed this gastroprotective effect. Administration of aminoguanidine did not influence the effect of sulfated-polysaccharide fraction. Our results suggest that sulfated-polysaccharide fraction exerts a protective effect against ethanol-induced gastric damage via activation of the NO/K ATP pathway.

  9. ATP as a biological hydrotrope.

    Science.gov (United States)

    Patel, Avinash; Malinovska, Liliana; Saha, Shambaditya; Wang, Jie; Alberti, Simon; Krishnan, Yamuna; Hyman, Anthony A

    2017-05-19

    Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells. Copyright © 2017, American Association for the Advancement of Science.

  10. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

    Science.gov (United States)

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W.; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  11. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts.

    Science.gov (United States)

    Shiozaki, Yuta; Sato, Masaki; Kimura, Maki; Sato, Toru; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2017-01-01

    ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1-P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K(+)-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K(+)-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K(+)-ATP-induced current were increased in solution without extracellular Ca(2+), but decreased in Na(+)-free extracellular solution. In the absence of both of extracellular Na(+) and Ca(2+), K(+)-ATP-induced currents were completely abolished. K(+)-ATP-induced Na(+) currents were inhibited by P2X7 inhibitor, while the Ca(2+) currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury.

  12. Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor.

    Science.gov (United States)

    Ozalp, Veli C; Pedersen, Tina R; Nielsen, Lise J; Olsen, Lars F

    2010-11-26

    Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

  13. Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP.

    Science.gov (United States)

    Dufour, Stéphane P; Patel, Rakesh P; Brandon, Angela; Teng, Xinjun; Pearson, James; Barker, Horace; Ali, Leena; Yuen, Ada H Y; Smolenski, Ryszard T; González-Alonso, José

    2010-12-01

    The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O(2)-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O(2) saturation (O(2)Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O(2)Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O(2)Hb (r(2) = 0.86-0.98; P = 0.01), decreases in venous plasma nitrite (r(2) = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r(2) = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r(2) = 0.59; P = 0.07). No relationship was observed with plasma ATP (r(2) = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature.

  14. Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP

    Science.gov (United States)

    Patel, Rakesh P.; Brandon, Angela; Teng, Xinjun; Pearson, James; Barker, Horace; Ali, Leena; Yuen, Ada H. Y.; Smolenski, Ryszard T.; González-Alonso, José

    2010-01-01

    The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O2-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O2 saturation (O2Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O2Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O2Hb (r2 = 0.86–0.98; P = 0.01), decreases in venous plasma nitrite (r2 = 0.93; P erythrocyte nitroso species (r2 = 0.74; P erythrocyte SNO (r2 = 0.59; P = 0.07). No relationship was observed with plasma ATP (r2 = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature. PMID:20852046

  15. Differential expression of ATP-dependent RNA helicase gene in ...

    African Journals Online (AJOL)

    AJL

    2012-02-07

    Feb 7, 2012 ... lysogeny broth (LB) medium at 4°C was induced for VBNC state; activated genes were detected using. mRNA differential display .... 191 amino acids. This cDNA sequence had a homology of 95 to 100% to the nucleotide of adenosine tripho- sphate (ATP)-dependent RNA helicase rh1B gene in different ...

  16. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  17. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  18. ATP Changes the Fluorescence Lifetime of Cyan Fluorescent protein via an Interaction with His148

    NARCIS (Netherlands)

    Borst, J.W.; Willemse, M.; Slijkhuis, R.; Krogt, G.; Laptenok, S.; Jalink, K.; Wieringa, B.; Fransen, J.A.M.

    2010-01-01

    Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent

  19. ATP changes the fluorescence lifetime of cyan fluorescent protein via an interaction with His148.

    NARCIS (Netherlands)

    Borst, J.W.; Willemse, M.P.; Slijkhuis, R.; Krogt, G. van der; Laptenok, S.P.; Jalink, K.; Wieringa, B.; Fransen, J.A.M.

    2010-01-01

    Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent

  20. Intracellular ATP concentration contributes to the cytotoxic and cytoprotective effects of adenosine.

    Directory of Open Access Journals (Sweden)

    Shujue Li

    Full Text Available Extracellular adenosine (ADE interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.

  1. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...

  2. ATP level and caffeine efficiency on cytokinesis inhibition in plants.

    Science.gov (United States)

    López-Sáez, J F; Mingo, R; González-Fernández, A

    1982-06-01

    Plant cytokinesis appears to be a topographically organized process of exocytosis. Golgi vesicles which contain cell wall precursors are translocated during telophase, by interzonal microtubules, to the equatorial region of the mitotic apparatus where they fuse with each other giving rise to the new cell wall. Caffeine inhibits cytokinesis by hindering Golgi vesicle coalescence. The present results demonstrate that treatments which increase the cellular ATP level (adenosine, cycloheximide and anisomycin) counteract caffein-induced cytokinesis inhibition in meristem cells of onion root tips (Allium cepa L.), while treatments which decrease ATP level potentiate this caffeine effect (dinitrophenol, fluoroacetate, low oxygen tensions, etc.). We postulate that caffeine, in competition with the cellular ATP level, blocks cell plate formation by inhibiting a certain ATPase activity required for membrane fusion of Golgi vesicles.

  3. ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses.

    Directory of Open Access Journals (Sweden)

    Shuang Wu

    Full Text Available The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

  4. Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

    Science.gov (United States)

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

    Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

  5. Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP.

    Science.gov (United States)

    Hogarth, D Kyle; Sandbo, Nathan; Taurin, Sebastien; Kolenko, Vladimir; Miano, Joseph M; Dulin, Nickolai O

    2004-08-01

    Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM-alpha-actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity.

  6. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants.

    Science.gov (United States)

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling roles in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor kinase), which is plant-specific. P2K1 (DORN1) is required for ATP-induced cellular responses (e.g., cytosolic Ca(2+) elevation, MAPK phosphorylation, and gene expression). Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of future research on extracellular ATP as a DAMP signal.

  7. The More Vivid the Imagination the Better: The Role of the Vividness of Imagination in Vasoconstriction Training and Vasodilatation Training.

    Science.gov (United States)

    Graef, Julia E; Rief, Winfried; Nestoriuc, Yvonne; Weise, Cornelia

    2017-12-01

    Blood volume pulse biofeedback represents an effective non-pharmacological treatment for migraine. However, the underlying mechanisms of blood volume pulse biofeedback are still unclear. This study investigated the influence of vividness of imagination, private body consciousness, perfectionism, and general self-efficacy on physiological (blood volume pulse amplitude) and psychological (session performance rated by participants and by trainers) success. Changes in skin conductance and skin temperature indicating habituation to training context were examined. Forty-five healthy male participants were randomized to four sessions of vasoconstriction training or vasodilatation training. Hierarchical linear models were estimated. Results showed significant changes of session performance rated by participants (UC = 0.62, p Vividness of imagination was highly important for both psychological achievement ratings (UC participants  = 1.3, p vividness of imagination.

  8. In Vivo Modelling of ATP1A3 G316S-Induced Ataxia in C. elegans Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects.

    Science.gov (United States)

    Sorkaç, Altar; Alcantara, Ivan C; Hart, Anne C

    2016-01-01

    The NIH Undiagnosed Diseases Program admitted a male patient with unclassifiable late-onset ataxia-like symptoms. Exome sequencing revealed a heterozygous de novo mutation converting glycine 316 to serine in ATP1A3, which might cause disease. ATP1A3 encodes the Na+/K+ ATPase pump α3-subunit. Using CRISPR/Cas9-mediated homologous recombination for genome editing, we modelled this putative disease-causing allele in Caenorhabditis elegans, recreating the patient amino acid change in eat-6, the orthologue of ATP1A3. The impact of the mutation on eat-6 function at the neuromuscular junction was examined using two behavioural assays: rate of pharyngeal pumping and sensitivity to aldicarb, a drug that causes paralysis over time via the inhibition of acetylcholinesterase. The patient allele decreased pumping rates and caused hypersensitivity to aldicarb. Animals heterozygous for the allele exhibited similar defects, whereas loss of function mutations in eat-6 were recessive. These results indicate that the mutation is dominant and impairs the neuromuscular function. Thus, we conclude that the de novo G316S mutation in ATP1A3 likely causes or contributes to patient symptoms. More broadly, we conclude that, for conserved genes, it is possible to rapidly and easily model human diseases in C. elegans using CRIPSR/Cas9 genome editing.

  9. In Vivo Modelling of ATP1A3 G316S-Induced Ataxia in C. elegans Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects.

    Directory of Open Access Journals (Sweden)

    Altar Sorkaç

    Full Text Available The NIH Undiagnosed Diseases Program admitted a male patient with unclassifiable late-onset ataxia-like symptoms. Exome sequencing revealed a heterozygous de novo mutation converting glycine 316 to serine in ATP1A3, which might cause disease. ATP1A3 encodes the Na+/K+ ATPase pump α3-subunit. Using CRISPR/Cas9-mediated homologous recombination for genome editing, we modelled this putative disease-causing allele in Caenorhabditis elegans, recreating the patient amino acid change in eat-6, the orthologue of ATP1A3. The impact of the mutation on eat-6 function at the neuromuscular junction was examined using two behavioural assays: rate of pharyngeal pumping and sensitivity to aldicarb, a drug that causes paralysis over time via the inhibition of acetylcholinesterase. The patient allele decreased pumping rates and caused hypersensitivity to aldicarb. Animals heterozygous for the allele exhibited similar defects, whereas loss of function mutations in eat-6 were recessive. These results indicate that the mutation is dominant and impairs the neuromuscular function. Thus, we conclude that the de novo G316S mutation in ATP1A3 likely causes or contributes to patient symptoms. More broadly, we conclude that, for conserved genes, it is possible to rapidly and easily model human diseases in C. elegans using CRIPSR/Cas9 genome editing.

  10. Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hayoz Sébastien

    2012-05-01

    Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

  11. Hydrogen peroxide lowers ATP levels in platelets without altering adenyalte energy charge and platelet function.

    Science.gov (United States)

    Holmsen, H; Robkin, L

    1977-03-10

    H2O2 irreversibly reduced metabolic platelet ATP levels with a corresponding accumulation of hypoxanthine. This process was enhanced by sodium azide or potassium cyanide and by increasing H2O2 concentrations. The adenylate energy charge was unaltered when less than two thirds of the metabolic ATP had disappeared but decreased markedly when more ATP disappeared. Platelet shape change, primary aggregation, dense granule and alpha-granule secretion were unaffected by H2O2-induced lowering of ATP provided that the adenylate energy charge did not fall by more than 5%; at greater adenylate energy charge reduction, platelet functions were inhibited. These results indicate that cell functions depend more on adenyalte energy charge than on the ATP level and expands the applicability of this view from bacterial systems to a mammalian cell, the human platelet.

  12. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.

    2008-01-01

    intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...... levcromakalim, pinacidil and P-1075. Experimental approach: mRNA and protein expression of K-ATP channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K-ATP channel drugs on rat MMA were studied in the genuine...... closed cranial window model and in myograph baths, respectively. Key results: Expression studies indicate that inwardly rectifying K+ (Kir)6.1/sulphonylurea receptor (SUR) 2B is the major K-ATP channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect...

  13. Two-step ATP-driven opening of cohesin head.

    Science.gov (United States)

    Marcos-Alcalde, Íñigo; Mendieta-Moreno, Jesús I; Puisac, Beatriz; Gil-Rodríguez, María Concepción; Hernández-Marcos, María; Soler-Polo, Diego; Ramos, Feliciano J; Ortega, José; Pié, Juan; Mendieta, Jesús; Gómez-Puertas, Paulino

    2017-06-12

    The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the "head" structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer.

  14. Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.

    Directory of Open Access Journals (Sweden)

    Kristian Parey

    Full Text Available ATP sulfurylase (ATPS catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2- with ATP to adenosine 5'-phosphosulfate (APS and pyrophosphate (PPi. In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

  15. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    Science.gov (United States)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

  16. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    Directory of Open Access Journals (Sweden)

    Elizabeth eMedina-Castellanos

    2014-11-01

    Full Text Available The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation. During this response, reactive oxygen species (ROS are produced by the NADPH oxidase (Nox1/NoxR complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK pathways by eATP, Ca2+ and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP. Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

  17. Glucose and the ATP paradox in yeast.

    NARCIS (Netherlands)

    Somsen, O.J.G.; Hoeben, M.A.; Esgalhado, M.E.L.M.; Snoep, J.L.; Visser, D.; van der Heijden, R.T.J.M.; Heijnen, J.J.; Westerhoff, H.V.

    2000-01-01

    A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be

  18. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    aliquots of standards increase quality control and ease daily operation. The medium (Lumin(PM) buffer, tap water or MilliQ water) for preparation of ATP-standard dilution significantly affected the rlu response of the ATP-standard dilutions (20% difference). The effect of dilution media and of sample...

  19. ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

    Science.gov (United States)

    Kwon, Hyuck Joon

    2013-01-01

    Skeletal patterns are prefigured by prechondrogenic condensation. Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. However, how morphogens regulate prechondrogenic condensation has remained unclear. Recently, it was demonstrated that synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. Thus, the present study has focused on whether ATP oscillations mediate the actions of major developmental morphogens such as FGF and Shh on prechondrogenic condensation. It has been shown that both FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. These results indicate that ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation. This study proposes that morphogens organize skeletal patterns via ATP oscillations. Copyright © 2012 John Wiley & Sons, Ltd.

  20. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Youn Ho; Lee, Seo Jin [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of); Jung, Junyang, E-mail: jjung@khu.ac.kr [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  1. ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

    Science.gov (United States)

    Kourie, J I

    1997-05-01

    Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl- channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl- channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, Imax and integral current, I') and kinetic parameters (frequency of opening FO, probability of the channel being open PO, mean open TO and closed Tc times) show a nonspecific block of the voltage- and Ca2+-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca2+ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg2+. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP4- > ADP3- > > > AMP2-. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP]cis and [Ca2+]cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca2+ between the SR and the sarcoplasm.

  2. A sensitive aptasensor for colorimetric detection of adenosine triphosphate based on the protective effect of ATP-aptamer complexes on unmodified gold nanoparticles.

    Science.gov (United States)

    Huo, Yuan; Qi, Liang; Lv, Xiao-Jun; Lai, Ting; Zhang, Jing; Zhang, Zhi-Qi

    2016-04-15

    Adenosine triphosphate (ATP) is the most direct source of energy in organisms. This study is the first to demonstrate that ATP-aptamer complexes provide greater protection for unmodified gold nanoparticles (AuNPs) against salt-induced aggregation than either aptamer or ATP alone. This protective effect was confirmed using transmission electron microscopy, dynamic light scattering, Zeta potential measurement, and fluorescence polarization techniques. Utilizing controlled particle aggregation/dispersion as a gauge, a sensitive and selective aptasensor for colorimetric detection of ATP was developed using ATP-binding aptamers as the identification element and unmodified AuNPs as the probe. This aptasensor exhibited a good linear relationship between the absorbance and the logarithm concentration of ATP within a 50-1000 nM range. ATP analogs such as guanosine triphosphate, uridine triphosphate and cytidine triphosphate resulted in little or no interference in the determination of ATP. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...... the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency...... for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A...

  4. Genetische Suppression endothelialer KCa3.1 und KCa2.3 unterdrückt die EDHF-vermittelte Vasodilatation und erzeugt Hypertonie

    OpenAIRE

    Brähler, Sebastian Paul

    2010-01-01

    Das Endothel nimmt Einfluss auf den Tonus der glatten Gefäßmuskulatur und ist somit wichtig für die adäquate Regulation der Gewebeperfusion und des systemischen Blutdrucks. Zur endothelvermittelten Vasodilatation tragen die diffusiblen Moleküle Stickstoffmonoxid (NO) und Prostazyklin (PGI2) sowie der sogenannte EDHF (endothelium-derived hyperpolarizing factor) bei. Obwohl dieser EDHF insbesondere in kleineren Arterien und...

  5. Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    The experiments concerned the effect of ethacrynic acid (0.5 mM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 microM). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose...

  6. ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.

    Science.gov (United States)

    O'Grady, Scott M; Patil, Nandadavi; Melkamu, Tamene; Maniak, Peter J; Lancto, Cheryl; Kita, Hirohito

    2013-09-15

      Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.

  7. Molecular devices for the regulation of chloroplast ATP synthase

    NARCIS (Netherlands)

    Hisabori, T.; Konno, H.; Ichimura, H.; Strotmann, H.; Bald, D.

    2002-01-01

    In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential

  8. Effect of inflammatory mediators on ATP release of human urothelial RT4 cells.

    Science.gov (United States)

    Mansfield, Kylie J; Hughes, Jessica R

    2014-01-01

    Inflammation is an important contributor to the aetiology of a number of bladder dysfunctions including interstitial cystitis, painful bladder syndrome, and overactive bladder. The aim of this study was to examine the effects of inflammatory mediators on urothelial ATP release. Human urothelial RT4 cells were exposed to normal buffer or varying concentrations of inflammatory mediators (bradykinin, histamine, and serotonin) in the presence or absence of hypotonic stretch stimuli (1 : 2 dilution of Krebs-Henseleit buffer). Others have demonstrated that bradykinin increased stretch-induced ATP release; however, we observed no change in control or stretch-induced ATP release with bradykinin. Pretreatment of RT4 cells with histamine or serotonin decreased stretch-induced ATP release (P = 0.037, P = 0.040, resp.). Previous studies have demonstrated increased ATP release in response to inflammation utilising whole bladder preparations in contrast to our simple model of cultured urothelial cells. The current study suggests that it is unlikely that there is a direct interaction between the release of inflammatory mediators and increased ATP release, but rather more complex interactions occurring in response to inflammation that lead to increased bladder sensation.

  9. Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels

    Directory of Open Access Journals (Sweden)

    See-Ziau Hoe

    2011-01-01

    Full Text Available INTRODUCTION: Gynura procumbens has been shown to decrease blood pressure via inhibition of the angiotensinconverting enzyme. However, other mechanisms that may contribute to the hypotensive effect have not been studied. OBJECTIVES: To investigate the cardiovascular effects of a butanolic fraction of Gynura procumbens in rats. METHODS: Anaesthetized rats were given intravenous bolus injections of butanolic fraction at doses of 2.5-20 mg/kg in vivo. The effect of butanolic fraction on vascular reactivity was recorded in isolated rat aortic rings in vitro. RESULTS: Intravenous administrations of butanolic fraction elicited significant (p<0.001 and dose-dependent decreases in the mean arterial pressure. However, a significant (p<0.05 decrease in the heart rate was observed only at the higher doses (10 and 20 mg/kg. In isolated preparations of rat aortic rings, phenylephrine (1×10-6 M- or potassium chloride (8×10-2 M-precontracted endothelium-intact and -denuded tissue; butanolic fraction (1×10-6-1×10-1 g/ml induced similar concentration-dependent relaxation of the vessels. In the presence of 2.5×10-3 and 5.0×10-3 g/ml butanolic fraction, the contractions induced by phenylephrine (1×10-9-3×10-5 M and potassium chloride (1×10-2-8×10-2 M were significantly antagonized. The calcium-induced vasocontractions (1×10-4-1×10-2 M were antagonized by butanolic fraction concentration-dependently in calcium-free and high potassium (6×10-2 M medium, as well as in calcium- and potassium-free medium containing 1×10-6 M phenylephrine. However, the contractions induced by noradrenaline (1×10-6 M and caffeine (4.5×10-2 M were not affected by butanolic fraction. CONCLUSION: Butanolic fraction contains putative hypotensive compounds that appear to inhibit calcium influx via receptor-operated and/or voltage-dependent calcium channels to cause vasodilation and a consequent fall in blood pressure.

  10. Fluid shifts, vasodilatation and ambulatory blood pressure reduction during long duration spaceflight

    DEFF Research Database (Denmark)

    Norsk, Peter; Asmar, Ali; Damgaard, Morten

    2015-01-01

    by 35–41% between 3 and 6 months on the International Space Station, which is more than during shorter flights.Twenty-four hour ambulatory brachial blood pressure is reduced by 8–10 mmHg by a decrease in systemic vascular resistance of 39%, which is not a result of the suppression of sympathetic nervous...... by astronauts on the International Space Station. ABSTRACT: Acute weightlessness in space induces a fluid shift leading to central volume expansion. Simultaneously, blood pressure is either unchanged or decreased slightly. Whether these effects persist for months in space is unclear. Twenty-four hour ambulatory...

  11. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Lenartowicz Malgorzata

    2016-08-01

    Full Text Available Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD or the rare autosomal disorder Wilson disease (WD, respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

  12. Taxol (paclitaxel) induces a detachment of phosphofructokinase from cytoskeleton of melanoma cells and decreases the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP.

    Science.gov (United States)

    Glass-Marmor, L; Beitner, R

    1999-04-09

    Glucose utilization through glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Here we report of a novel mechanism of action of taxol (paclitaxel; Baccatin III N-benzyl-beta-phenylisoserine ester), the anti-microtubule agent with remarkable anticancer activity. We show that taxol affects both levels of regulation of glycolysis in melanoma cells; it decreases the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis, and also causes a detachment of phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis, from the cytoskeleton of B16 melanoma cells. These effects of taxol were dose-dependent, and preceded the decrease in ATP levels and cell viability. Thus, taxol not only inhibits the essential dynamic processes of microtubule network, but also reduces glycolysis, through the novel mechanisms described here.

  13. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

    Directory of Open Access Journals (Sweden)

    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

  14. Novel selective phosphodiesterase type 1 inhibitors cause vasodilatation and lower blood pressure in rats

    DEFF Research Database (Denmark)

    Laursen, Morten; Thinggaard, Lilliana Beck; Kehler, Jan

    2017-01-01

    BACKGROUND AND PURPOSE: The PDE enzymes (PDE1-11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027...... and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated...... mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration-dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ, and adenylate cyclase, SQ22536, and in preparations without endothelium. In anaesthetized rats, Lu AF41228...

  15. Redox regulation of ATP sulfurylase in microalgae

    Czech Academy of Sciences Publication Activity Database

    Prioretti, L.; Lebrun, R.; Gontero, B.; Giordano, Mario

    2016-01-01

    Roč. 478, č. 4 (2016), s. 1555-1562 ISSN 0006-291X Institutional support: RVO:61388971 Keywords : ATP sulfurylase * cysteine * Sulfur metabolism Subject RIV: EE - Microbiology, Virology Impact factor: 2.466, year: 2016

  16. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  17. Vasculoprotective and vasodilatation effects of herbal formula (Sahatsatara) and piperine in spontaneously hypertensive rats.

    Science.gov (United States)

    Booranasubkajorn, Suksalin; Huabprasert, Sukit; Wattanarangsan, Jantanee; Chotitham, Pruksa; Jutasompakorn, Pinpilai; Laohapand, Tawee; Akarasereenont, Pravit; Tripatara, Pinpat

    2017-01-15

    The herbal formula (Sahatsatara, STF), the Thai traditional poly-herbal recipe, has been used for treatment of muscle pain, anti-flatulence and numbness on hands and feet, with the caution when used in hypertensive patients. However, there is no scientific evidence to prove its effects on cardiovascular system. Piperine is the proposed major active compound in STF. It is shown to have antihypertensive effect in the L-NAME-induced endothelial dysfunction rats. This study investigated the pharmacokinetics, mechanism of action, as well as the hemodynamic and vasoactive effect and toxicity of STF and piperine using spontaneously hypertensive rats (SHR) and normal Wistar rats (NWR). The amount of piperine in STF was measured by ultra performance liquid chromatography (UPLC). SHR and NWR were gavaged with piperine (50mg/kg/day) or STF (100, 300, or 1000mg/kg/day) alone or together with L-NAME (in drinking water) for 28 days. Hemodynamic effects were monitored by noninvasive tail cuff every 7 days. Vasorelaxation effect on the thoracic aorta in organ chamber was observed through force transducer at the end of the experiment. Biochemical parameters for kidney and liver toxicity were measured. In addition, pharmacokinetic study was performed using non-compartment analysis. The amount of piperine in STF was 1.29%w/w. Both STF and piperine did not affect blood pressure and heart rate in both SHR and NWR. Interestingly, STF and piperine increased acetylcholine-induced vasorelaxation of isolated thoracic aorta and have vascoluprotective effect in nitric oxide (NO) impaired rats. No liver or kidney toxicity was found in this study. Non-compartment pharmacokinetic analysis showed that the time to reach maximum concentration (Tmax) of plasma piperine after administration of piperine and STF were 3.9 and 1.7h, respectively. This result suggested that piperine in the recipe had better absorption than the pure standard piperine. STF had no effect on blood pressure in both SHR and NWR

  18. Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP

    Directory of Open Access Journals (Sweden)

    Ruojia Zhu

    2017-09-01

    Full Text Available Extracellular ATP (eATP has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein α subunit (Gα (gpa1-1 and gpa1-2 and enhanced in Gα-over-expression (OE lines (wGα and cGα. Ethylenebis(oxyethylenenitrilo tetraacetic acid (EGTA and Gd3+ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca2+ influx was impaired in Gα null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in Gα null mutants. In addition, some eATP-induced genes' expression was also impaired in Gα null mutants. Based on these results, we propose that heterotrimeric Gα-regulated Ca2+ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.

  19. Sildenafil citrate (Viagra) enhances vasodilatation by atrial natriuretic peptide in normal dogs.

    Science.gov (United States)

    Ishikura, Fuminobu; Beppu, Shintaro; Asanuma, Toshihiko; Seward, James B; Khandheria, Bijoy K

    2007-12-01

    Sildenafil citrate (Viagra) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5, which might enhance the vasorelaxant and natriuretic actions of atrial natriuretic peptide (ANP) in patients with heart failure. The objective of this study was to examine the combined effect of Viagra on hemodynamic changes during infusion of exogenous ANP. Healthy male beagles were used to assess systemic blood pressure, pulmonary artery pressure (PAP), and plasma levels of cGMP. After hemodynamic variables were measured, 0.1 microg.kg(-1).min(-1) of ANP was given during this study. One hour after initiating infusion of ANP, 2 mg/kg of sildenafil citrate or vehicle was given orally via a nasogastric tube. Hemodynamic changes were measured before and 1 h after these administrations. Mean systemic and PAP decreased during infusion of ANP, and further decreased after sildenafil citrate administration, however, mean systemic blood pressure decreased within 10 mmHg. Plasma levels of cGMP also increased after sildenafil citrate administration. In normal dogs, sildenafil citrate enhances the vasodilator effect of ANP by increasing the cGMP level, however, the concomitant use of sildenafil citrate with ANP will not induce severe hypotension.

  20. Association of the α(2)δ(1) subunit with Ca(v)3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes.

    Science.gov (United States)

    Thompson, William R; Majid, Amber S; Czymmek, Kirk J; Ruff, Albert L; García, Jesús; Duncan, Randall L; Farach-Carson, Mary C

    2011-09-01

    Voltage-sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α(1) pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type α(1) subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the α(2)δ auxiliary subunit on T-type VSCCs and the physiologic consequences of that association are incompletely understood and have yet to be investigated in bone. In this study we postulated that the auxiliary α(2) δ subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca(v) 3.2 (α(1H) ) subunit. We demonstrated by reverse-transcriptase polymerase chain reaction, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type Ca(v)3.2(α(1H)) subunit more abundantly than the L-type Ca(v)1.2 (α(1C)) subunit. We also demonstrated that the α(2) δ(1) subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca(v)3.2 (α(1H)) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of α(2) δ(1) completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α(2)δ(1) subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α(2)δ(1) knockdown showed decreased membrane localization of Ca(v) 3.2 (α(1H)) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Ca(v) 3.2 (α(1H)) at the cell membrane. Copyright © 2011 American Society for Bone and Mineral Research.

  1. The glycolytic flux in Escherichia coli is controlled by the demand for ATP

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

    2002-01-01

    of the membrane-bound (F1F0) H+-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing......The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F-1 part...... additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (> 75%) resides...

  2. ATP-regenerating system in the cilia of Paramecium caudatum.

    Science.gov (United States)

    Noguchi, M; Sawada, T; Akazawa, T

    2001-03-01

    The energy supply for eukaryotic ciliary and flagellar movement is thought to be maintained by ATP-regenerating enzymes such as adenylate kinase, creatine kinase and arginine kinase. In this study, the energy-supplying system for the ciliary movement of Paramecium caudatum was examined. Arginine kinase and adenylate kinase activities were detected in the cilia. To demonstrate that phosphoarginine satisfactorily supplies high-energy phosphate compounds into the narrow ciliary space, we prepared an intact ciliated cortical sheet from live Paramecium caudatum. These cortical sheets, with an intact ciliary membrane, produced a half-closed system in which each cilium was covered with a ciliary membrane with an opening to the cell body. Ciliary beating on the intact cortical sheets was induced by perfusing not only ATP but also ADP. Addition of phosphoarginine (0.2 mmol l(-1)) increased the beat frequency. A further increase in beat frequency was observed in 0.4 mmol l(-1) phosphoarginine, and this was enhanced when the cilia were reactivated with relatively low concentrations of ATP. We have demonstrated that phosphoarginine supplies energy as a 'phosphagen' for ciliary beating in Paramecium caudatum, suggesting that phosphoarginine functions not only as a reservoir of energy but also as a transporter of energy in these continuously energy-consuming circumstances. http://www.biologists.com/JEB/movies/jeb3123.html

  3. Extracellular ATP is a key modulator of alveolar bone loss in periodontitis.

    Science.gov (United States)

    Binderman, Itzhak; Gadban, Nasir; Yaffe, Avinoam

    2017-09-01

    Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca+2. Increased extracellular ATP (eATP) by interacting with P2×7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3-inflammasome activation via P2×7. Then, the inflammatory cells secrete cytokines, interleukin IL-1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery

    Science.gov (United States)

    Sato, Takato; Ohnuki, Jun; Takano, Mitsunori

    2017-12-01

    A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

  5. Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery.

    Science.gov (United States)

    Sato, Takato; Ohnuki, Jun; Takano, Mitsunori

    2017-12-07

    A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

  6. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    Science.gov (United States)

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-09

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  8. ATP8B1 and ATP11C: Two Lipid Flippases Important for Hepatocyte Function

    NARCIS (Netherlands)

    Naik, Jyoti; de Waart, Dirk R.; Utsunomiya, Karina; Duijst, Suzanne; Mok, Kam Ho; Oude Elferink, Ronald P. J.; Bosma, Piter J.; Paulusma, Coen C.

    2015-01-01

    P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe

  9. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid...

  10. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  11. Voltage Dependence of ATP Secretion in Mammalian Taste Cells

    Science.gov (United States)

    Romanov, Roman A.; Rogachevskaja, Olga A.; Khokhlov, Alexander A.; Kolesnikov, Stanislav S.

    2008-01-01

    Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels. PMID:19029378

  12. K-ATP channels promote the differential degeneration of dopaminergic midbrain neurons.

    Science.gov (United States)

    Liss, Birgit; Haeckel, Olga; Wildmann, Johannes; Miki, Takashi; Seino, Susumu; Roeper, Jochen

    2005-12-01

    The selective degeneration of dopaminergic (DA) midbrain neurons in the substantia nigra (SN) is a hallmark of Parkinson disease. DA neurons in the neighboring ventral tegmental area (VTA) are significantly less affected. The mechanisms for this differential vulnerability of DA neurons are unknown. We identified selective activation of ATP-sensitive potassium (K-ATP) channels as a potential mechanism. We show that in response to parkinsonism-inducing toxins, electrophysiological activity of SN DA neurons, but not VTA DA neurons, is lost owing to activation of K-ATP channels. This selective K-ATP channel activation is controlled by differences in mitochondrial uncoupling between SN and VTA DA neurons. Genetic inactivation of the K-ATP channel pore-forming subunit Kir6.2 resulted in a selective rescue of SN but not VTA DA neurons in two mechanistically distinct mouse models of dopaminergic degeneration, the neurotoxicological 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model and the mutant weaver mouse. Thus, K-ATP channel activation has an unexpected role in promoting death of DA neurons in chronic disease.

  13. A cytotoxic Petiveria alliacea dry extract induces ATP depletion and decreases β-F1-ATPase expression in breast cancer cells and promotes survival in tumor-bearing mice

    Directory of Open Access Journals (Sweden)

    John F. Hernández

    Full Text Available Abstract Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model.

  14. Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts.

    Science.gov (United States)

    Malik Ghulam, Mustafa; Zghidi-Abouzid, Ouafa; Lambert, Emeline; Lerbs-Mache, Silva; Merendino, Livia

    2012-06-01

    The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

  15. ATP and astrocytes play a prominent role in the control of the respiratory pattern generator in the lamprey.

    Science.gov (United States)

    Cinelli, Elenia; Iovino, Ludovica; Mutolo, Donatella

    2017-12-01

    The paratrigeminal respiratory group (pTRG) is responsible for the respiratory pattern generation in the lamprey. The role of ATP and astrocytes, known to control respiratory activity in mammals, was investigated in the lamprey respiratory network. ATP microinjected into the pTRG induces a biphasic response consisting of marked increases in respiratory frequency mediated by P2X receptors followed by a decrease in the respiratory motor output due to the ATP metabolite adenosine. We provide evidence that astrocytes are involved in the genesis of the normal respiratory pattern, ATP-induced responses and acidification-induced increases of the respiratory activity. The function of astrocytes in rhythmic networks appears to be phylogenetically conserved. The role of ATP and astrocytes in respiratory rhythm modulation has been recently investigated in neonatal rodents. However, no information on the role of ATP and astrocytes within the respiratory network of the lamprey is available, particularly within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator. To address these issues, the present study was carried out on isolated brainstems of the adult lamprey. Bath application of ATP caused marked increases in respiratory frequency followed by decreases in the respiratory motor output, mediated by the ATP metabolite adenosine at the level of the pTRG. Bath applications and microinjections of agonists and antagonists of purinergic receptors showed that ATP increased respiratory activity through an action on pTRG P2X receptors. To disclose the respiratory role of astrocytes, we used bath application of the gliotoxin aminoadipic acid, which dramatically depressed the respiratory motor output that, however, promptly recovered following glutamine application. Furthermore, the excitatory responses to ATP-γ-S (a non-hydrolysable ATP analogue), but not to substance P, microinjected into the pTRG, were abolished. Finally, we also

  16. Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.

    Science.gov (United States)

    Akopova, Irina; Tatur, Sabina; Grygorczyk, Mariusz; Luchowski, Rafał; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Grygorczyk, Ryszard

    2012-03-01

    Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

  17. Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast.

    Science.gov (United States)

    Niedzwiecka, Katarzyna; Tisi, Renata; Penna, Sara; Lichocka, Malgorzata; Plochocka, Danuta; Kucharczyk, Roza

    2018-01-01

    The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia

    Science.gov (United States)

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W.; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-01-01

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. PMID:27739425

  19. Distinct Conformation of ATP Molecule in Solution and on Protein.

    Science.gov (United States)

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2' exo or C2' endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule.

  20. High glucose impairs ATP formation on the surface of human peripheral blood B lymphocytes.

    Science.gov (United States)

    Sakowicz-Burkiewicz, Monika; Grden, Marzena; Maciejewska, Izabela; Szutowicz, Andrzej; Pawelczyk, Tadeusz

    2013-07-01

    Diabetes-associated lymphocyte dysfunction may be attributed to the direct effect of hyperglycemia, but the impact of glucose concentration on B cell functionality is not fully resolved. Since, adenosine 5'-triphosphate (ATP) and its metabolite adenosine are the core constituents of the purinergic signaling network involved in regulation of immune response we aimed to investigate the impact of high glucose concentration on ATP outflow and metabolism on B cell surface. Purified human peripheral blood B cells cultured at high glucose (25 mM) concentration released significantly less ATP (~60%) comparing to cells cultured in low glucose (5mM) concentration. We observed that high glucose altered ATP hydrolysis on B cell surface due to increased activity of nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39). In the presence of 10 μM [(3)H]AMP and 100 μM ATP significant quantities of [(3)H]ADP and [(3)H]ATP were generated, although the AMP to ADP phosphorylation potential of B cells cultured in high glucose decreased significantly. The flow cytometry analysis revealed that the level of ecto-adenylate kinase 1β (AK1β) on surface of B cells cultured in high glucose decreased significantly. Inhibition of NTPDase1/CD39 activity with 100 μM ARL67156 resulted in decreased cell viability, although significantly more viable cells retained in the culture media containing low glucose compared to high glucose media. Selective inhibition of P2X7 purinergic receptor irrespective of glucose concentration completely protected B cells against the ARL 67156-induced cell death. We assume that high glucose-induced alteration of ATP handling on B cell surface might contribute to impaired functionality of B cells in diabetes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Electric field driven torque in ATP synthase.

    Directory of Open Access Journals (Sweden)

    John H Miller

    Full Text Available FO-ATP synthase (FO is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1 overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

  2. The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

    Directory of Open Access Journals (Sweden)

    Manuela Antoniel

    2014-04-01

    Full Text Available The oligomycin-sensitivity conferring protein (OSCP of the mitochondrial FOF1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between FO and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP, whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca2+ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca2+-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s that switches the enzyme of life in a channel able to precipitate cell death.

  3. ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells

    NARCIS (Netherlands)

    C. Burkard (Christine); M.H. Verheije (Monique); B.L. Haagmans (Bart); F.J.M. van Kuppeveld (Frank ); P.J.M. Rottier (Peter); B.J. Bosch (Berend Jan); C.A.M. de Haan (Cornelis)

    2015-01-01

    textabstractIn addition to transporting ions, the multisubunit Na+,K+-ATPase also functions by relaying cardiotonic steroid (CTS)-binding- induced signals into cells. In this study, we analyzed the role of Na+,K+-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection.

  4. ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells

    NARCIS (Netherlands)

    Burkard, Christine; Verheije, Monique H; Haagmans, Bart L; van Kuppeveld, Frank J; Rottier, Peter J M; Bosch, Berend-Jan; de Haan, Cornelis A M

    2015-01-01

    Besides by transporting ions the multi-subunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid-binding induced signals into cells. In this study we analyzed the role of Na(+),K(+)-ATPase and in particular of its ATP1A1 α-subunit during coronavirus (CoV) infection. As controls, the

  5. A Label-Free Luminescent Switch-On Assay for ATP Using a G-Quadruplex-Selective Iridium(III) Complex

    OpenAIRE

    Ka-Ho Leung; Lihua Lu; Modi Wang; Tsun-Yin Mak; Daniel Shiu-Hin Chan; Fung-Kit Tang; Chung-Hang Leung; Hiu-Yee Kwan; Zhiling Yu; Dik-Lung Ma

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5'-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal vio...

  6. Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

    Science.gov (United States)

    Spaans, F; Melgert, B N; Chiang, C; Borghuis, T; Klok, P A; de Vos, P; van Goor, H; Bakker, W W; Faas, M M

    2014-08-01

    Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

  8. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, ou...

  9. Limitations of ATP as a measure of microbial biomass | Stuart ...

    African Journals Online (AJOL)

    Estimates of the total living biomass of micro-organisms on decomposing kelp detritus, calculated indirectly from the concentration of ATP, were compared with those obtained directly from cell numbers and volumes. Large overestimates in biomass were obtained from ATP x 250, and C:ATP ratios varied considerably with ...

  10. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Masaki Nakano

    2017-08-01

    Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

  11. MicroRNA-320a and microRNA-4496 attenuate Helicobacter pylori cytotoxin-associated gene A (CagA)-induced cancer-initiating potential and chemoresistance by targeting β-catenin and ATP-binding cassette, subfamily G, member 2.

    Science.gov (United States)

    Kang, Dong Woo; Yang, Eun Sun; Noh, Yu Na; Hwang, Won Chan; Jo, Se-Young; Suh, Young-Ah; Park, Won Sang; Choi, Kang-Yell; Min, Do Sik

    2017-04-01

    Infection with Helicobacter pylori is closely linked to an increased risk of gastric cancer. Although cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is known to be a causal factor for gastric carcinogenesis, the molecular link between CagA and gastric cancer-initiating cell (CIC)-like properties remains elusive. Here, we demonstrate that CagA is required for increased expression of β-catenin and its target CIC markers via downregulation of microRNA (miR)-320a and miR-4496. CagA promoted gastric CIC properties and was responsible for chemoresistance. miR-320a and miR-4496 attenuated the in vitro self-renewal and tumour-initiating capacity of CagA-expressing CICs by targeting β-catenin. Moreover, miR-320a and miR-4496 decreased CagA-induced chemoresistance by targeting ATP-binding cassette, subfamily G, member 2 (ABCG2) at the transcriptional and post-transcriptional levels, respectively. Combination therapy with 5-fluorouracil and miR-320a/miR-4496 suppressed gastric tumourigenesis and metastatic potential in an orthotopic mouse model, probably via suppression of CagA-induced CIC properties and chemoresistance. Our results provide novel evidence that CIC properties, chemoresistance and tumourigenesis associated with H. pylori are linked to CagA-induced upregulation of β-catenin and ABCG2. These data provide novel insights into the molecular mechanisms of CagA-induced carcinogenisis and the therapeutic potential of of miR-320a and miR-4496. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  12. How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

    Science.gov (United States)

    Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

    2013-07-01

    A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

  13. Migraine can be induced by sildenafil without changes in middle cerebral artery diameter

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Thomsen, Lars Lykke; Birk, Steffen

    2003-01-01

    GMP)-mediated vasodilatation. We examined whether sildenafil (Viagra), a selective inhibitor of cGMP-hydrolysing phosphodiesterase 5 (PDE5), which acts exclusively by increasing cGMP, can induce migraine and dilatation of cerebral arteries. We included 12 patients with migraine without aura in this double-blind, placebo...

  14. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  15. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-01-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  16. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  17. Anesthetic Propofol Overdose Causes Vascular Hyperpermeability by Reducing Endothelial Glycocalyx and ATP Production

    Science.gov (United States)

    Lin, Ming-Chung; Lin, Chiou-Feng; Li, Chien-Feng; Sun, Ding-Ping; Wang, Li-Yun; Hsing, Chung-Hsi

    2015-01-01

    Prolonged treatment with a large dose of propofol may cause diffuse cellular cytotoxicity; however, the detailed underlying mechanism remains unclear, particularly in vascular endothelial cells. Previous studies showed that a propofol overdose induces endothelial injury and vascular barrier dysfunction. Regarding the important role of endothelial glycocalyx on the maintenance of vascular barrier integrity, we therefore hypothesized that a propofol overdose-induced endothelial barrier dysfunction is caused by impaired endothelial glycocalyx. In vivo, we intraperitoneally injected ICR mice with overdosed propofol, and the results showed that a propofol overdose significantly induced systemic vascular hyperpermeability and reduced the expression of endothelial glycocalyx, syndecan-1, syndecan-4, perlecan mRNA and heparan sulfate (HS) in the vessels of multiple organs. In vitro, a propofol overdose reduced the expression of syndecan-1, syndecan-4, perlecan, glypican-1 mRNA and HS and induced significant decreases in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio and ATP concentrations in human microvascular endothelial cells (HMEC-1). Oligomycin treatment also induced significant decreases in the NAD+/NADH ratio, in ATP concentrations and in syndecan-4, perlecan and glypican-1 mRNA expression in HMEC-1 cells. These results demonstrate that a propofol overdose induces a partially ATP-dependent reduction of endothelial glycocalyx expression and consequently leads to vascular hyperpermeability due to the loss of endothelial barrier functions. PMID:26023717

  18. ATP synthases from archaea: the beauty of a molecular motor.

    Science.gov (United States)

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants1[OPEN

    Science.gov (United States)

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-01-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. PMID:26224801

  20. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

    Science.gov (United States)

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-09-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg(2+) as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. © 2015 American Society of Plant Biologists. All Rights Reserved.

  1. Interaction of diazoxide and cromakalim with ATP-regulated K+ channels in rodent and clonal insulin-secreting cells.

    Science.gov (United States)

    Jaggar, J H; Harding, E A; Ayton, B J; Dunne, M J

    1993-02-01

    The hyperglycaemia-inducing sulphonamide diazoxide has been previously shown to mediate its effects upon insulin secretion by opening K+ channels and hyperpolarizing the beta-cell membrane. The target site has been characterized as the ATP-regulated K+ (K+ATP) channel protein. In the present study, a detailed investigation of interactions of diazoxide and another K+ channel opener, cromakalim, with K+ATP channels has been performed in individual insulin-secreting cells using patch-clamp techniques. In agreement with previous studies, diazoxide and cromakalim were found to be effective only when ATP was present upon the inside face of the plasma membrane. The ability of both diazoxide and cromakalim to open channels was, however, found to diminish with time following isolation of inside-out patches. Within seconds of forming the recording configuration, the actions of both compounds were potent, and were found to decline steadily as the number of operational channels decreased ('run-down'). In open cells, where the plasma membrane remains partially intact, the rate of run-down was significantly reduced, and effects of channel openers were recorded up to 80 min following cell permeabilization. We also demonstrated that in the absence of ATP, but in the presence of ADP, both diazoxide and cromakalim were able to open K+ATP channels. Interestingly, once the effects of diazoxide and cromakalim on K+ATP channels in the presence of ATP were lost, both compounds opened channels in the presence of ADP. One implication of these data is that the actions of diazoxide and cromakalim involve regulatory proteins associated with the ion channel; this molecule is able to bind ATP, ADP and possibly other cytosolic nucleotides.

  2. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System

    Directory of Open Access Journals (Sweden)

    Hyuck Joon Kwon

    2015-01-01

    Full Text Available Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  3. OS053. Trophoblast invasion in the mesometrial triangle of atp infused pregnant rats.

    Science.gov (United States)

    Spaans, F; Chiang, C W; Melgert, B N; Klok, P A; Borghuis, T; Bakker, W W; Faas, M M

    2012-07-01

    Poor placentation (disturbed and decreased trophoblast invasion) is a hallmark of preeclampsia (PE), which is a major complication of pregnancy. Unfortunately, the cause and mechanism of disturbed trophoblast invasion is still unknown. The pro-inflammatory agent ATP has been shown to induce PE-like signs, after a single infusion in pregnant rats. These PE-like characteristics include proteinuria and decreased fetal weight. Since purinergic ATP receptors are expressed on trophoblast cells, we aimed to study the effect of ATP infusion on trophoblast invasion in pregnant rats in this pilot study. Pregnant rats received a single ATP (n=4) or saline (control,ni=5) infusion via a permanent jugular vein cannula on day 14 of pregnancy. At the time of maximal trophoblast invasion (day 17 of pregnancy) rats were sacrificed and placentas with mesometrial triangle were collected, fixed in zinc-buffer and embedded in paraffin. 4 μm sections were stained with monoclonal α-cytokeratin antibodies. In the mesometrial triangle, the maternal part of the rat placenta, the percentage of surface area of trophoblast invasion was evaluated using computerized image analysis. Also, the depth and width of invasion were analyzed by subdividing the mesometrial triangle in three concentric depth levels of equal width. In addition, trophoblast invaded versus non-invaded spiral arteries in the mesometrial triangle were quantified. In the mesometrial triangle, no changes in percentage of surface area of trophoblast invasion and percentage of invaded spiral arteries were observed after ATP infusion. However, the pattern of trophoblast invasion appeared to be disturbed in ATP infused rats, with a decreased depth of invasion and an increased width of invasion, resulting in a trend towards a decreased depth/width ratio of trophoblast invasion in ATP infused rats. In this (pilot) study we showed an altered trophoblast invasion pattern in the mesometrial triangle of the placenta, although no

  4. Functional K(ATP) channels in the rat retinal microvasculature: topographical distribution, redox regulation, spermine modulation and diabetic alteration.

    Science.gov (United States)

    Ishizaki, Eisuke; Fukumoto, Masanori; Puro, Donald G

    2009-05-15

    The essential task of the circulatory system is to match blood flow to local metabolic demand. However, much remains to be learned about this process. To better understand how local perfusion is regulated, we focused on the functional organization of the retinal microvasculature, which is particularly well adapted for the local control of perfusion. Here, we assessed the distribution and regulation of functional K(ATP) channels whose activation mediates the hyperpolarization induced by adenosine. Using microvascular complexes freshly isolated from the rat retina, we found a topographical heterogeneity in the distribution of functional K(ATP) channels; capillaries generate most of the K(ATP) current. The initiation of K(ATP)-induced responses in the capillaries supports the concept that the regulation of retinal perfusion is highly decentralized. Additional study revealed that microvascular K(ATP) channels are redox sensitive, with oxidants increasing their activity. Furthermore, the oxidant-mediated activation of these channels is driven by the polyamine spermine, whose catabolism produces oxidants. In addition, our observation that spermine-dependent oxidation occurs predominately in the capillaries accounts for why they generate most of the K(ATP) current detected in retinal microvascular complexes. Here, we also analysed retinal microvessels of streptozotocin-injected rats. We found that soon after the onset of diabetes, an increase in spermine-dependent oxidation at proximal microvascular sites boosts their K(ATP) current and thereby virtually eliminates the topographical heterogeneity of functional K(ATP) channels. We conclude that spermine-dependent oxidation is a previously unrecognized mechanism by which this polyamine modulates ion channels; in addition to a physiological role, spermine-dependent oxidation may also contribute to microvascular dysfunction in the diabetic retina.

  5. A taste for ATP: neurotransmission in taste buds

    Science.gov (United States)

    Kinnamon, Sue C.; Finger, Thomas E.

    2013-01-01

    Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

  6. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    Science.gov (United States)

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  7. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking.

    Science.gov (United States)

    Yi, Ling; Kaler, Stephen G

    2015-05-01

    ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7A(P1386S) causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7A(P1386S) partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  8. Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    JoAnn S. Roberts

    2017-07-01

    Full Text Available Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs. P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP/P2X7 signaling, such as the generation of reactive oxygen species (ROS via the mitochondria and NADPH oxidases (NOX from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05. Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1 during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05 in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked

  9. Mitochondrial ATP Depletion Disrupts Caco-2 Monolayer Integrity and Internalizes Claudin 7.

    Science.gov (United States)

    JanssenDuijghuijsen, Lonneke M; Grefte, Sander; de Boer, Vincent C J; Zeper, Lara; van Dartel, Dorien A M; van der Stelt, Inge; Bekkenkamp-Grovenstein, Melissa; van Norren, Klaske; Wichers, Harry J; Keijer, Jaap

    2017-01-01

    Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the

  10. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III) complex.

    Science.gov (United States)

    Leung, Ka-Ho; Lu, Lihua; Wang, Modi; Mak, Tsun-Yin; Chan, Daniel Shiu-Hin; Tang, Fung-Kit; Leung, Chung-Hang; Kwan, Hiu-Yee; Yu, Zhiling; Ma, Dik-Lung

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5'-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  11. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III complex.

    Directory of Open Access Journals (Sweden)

    Ka-Ho Leung

    Full Text Available We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III complex for the detection of adenosine-5'-triphosphate (ATP in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  12. The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin.

    Science.gov (United States)

    Kardos, Roland; Pozsonyi, Kinga; Nevalainen, Elisa; Lappalainen, Pekka; Nyitrai, Miklós; Hild, Gábor

    2009-03-18

    Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.

  13. Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

    KAUST Repository

    Beke-Somfai, T.

    2013-01-23

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

  14. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  15. MRT letter: Expression of ATP sensor protein in Caenorhabditis elegans.

    Science.gov (United States)

    Kishikawa, Jun-ichi; Fujikawa, Makoto; Imamura, Hiromi; Yasuda, Kayo; Noji, Hiroyuki; Ishii, Naoaki; Mitani, Shohei; Yokoyama, Ken

    2012-01-01

    Adenosine 5'-triphosphate (ATP) is the major energy currency and is involved in many biological processes. The ATP-monitoring system for cells in animals can be helpful to study the relationship between energy metabolism and biological processes. The fluorescent ATP biosensor ATeam (ATP indicator based on Epsilon subunit for Analytical Measurements), which has been reported to monitor ATP levels in cultured cells on the basis of fluorescence resonance energy transfer (FRET), was introduced into nematodes by microinjection and UV-irradiation method. To confirm whether ATeam functions as an ATP sensor in nematode cells, the authors measured FRET of ATeam in cells of transgenic nematode. The ATeam was expressed in target cells in nematode. In vulva cells, ATP levels in the cytosol were higher than those in mitochondria. ATeam also sensed ATP level change in cultured cells from the transgenic nematode. These experiments indicated that ATeam is available for detection of changes in ATP levels in nematode cells. Copyright © 2011 Wiley Periodicals, Inc.

  16. Orientation and mobility of actin in different intermediate states of the ATP hydrolysis cycle.

    Science.gov (United States)

    Khaimina, S S; Wrzosek, A; Dabrowska, R; Borovikov, Yu S

    2005-10-01

    Using polarization fluorimetry, we have investigated conformational changes of FITC-phalloidin-labeled F-actin in ghost muscle fibers. These changes were induced by myosin subfragment-1 (S1) in the absence and presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes of orientation and mobility of actin monomers during the ATPase cycle. The most pronounced differences in orientation (~4 degrees ) and in mobility (~43%) of actin were found between the actomyosin states induced by MgADP and MgATP.

  17. The DNA gyrase-quinolone complex. ATP hydrolysis and the mechanism of DNA cleavage

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis......, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606-22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence...... of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA...

  18. A polycystin-type transient receptor potential (Trp channel that is activated by ATP

    Directory of Open Access Journals (Sweden)

    David Traynor

    2017-02-01

    Full Text Available ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.

  19. Glycolytic ATP fuels the plasma membrane calcium pump critical for pancreatic cancer cell survival.

    Science.gov (United States)

    James, Andrew D; Chan, Anthony; Erice, Oihane; Siriwardena, Ajith K; Bruce, Jason I E

    2013-12-13

    Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca(2+) ([Ca(2+)]i), as the ATP-dependent plasma membrane Ca(2+) ATPase (PMCA) is critical for maintaining low [Ca(2+)]i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca(2+)]i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca(2+)]i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.

  20. L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

    KAUST Repository

    Jourdain, P.

    2016-02-19

    Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

  1. Role of Erythrocyte-released ATP in the Regulation of Microvascular Oxygen Supply in Skeletal Muscle

    Science.gov (United States)

    Ellsworth, Mary L.; Ellis, Christopher G.; Sprague, Randy S.

    2015-01-01

    In a 1914 book entitled The Respiratory Function of the Blood, Joseph Barcroft stated that “the cell takes what it needs and leaves the rest.” He postulated that there must be both a “call for oxygen” and a “mechanism by which the call elicits a response…” In the past century, intensive investigation has provided significant insights into the hemodynamic and biophysical mechanisms involved in supplying oxygen to skeletal muscle. However, the identification of the mechanism by which tissue oxygen needs are sensed and the affector responsible for altering the upstream vasculature to enable the need to be appropriately met has been a challenge. In 1995, Ellsworth et al proposed that the oxygen carrying erythrocyte, by virtue of its capacity to release the vasoactive mediator ATP in response to a decrease in oxygen saturation, could serve both roles. Several in vitro and in situ studies have established that exposure of erythrocytes to reduced oxygen tension induces the release of ATP which does result in a conducted arteriolar vasodilation with a sufficiently rapid time course to make the mechanism physiologically relevant. The components of the signaling pathway for the controlled release of ATP from erythrocytes in response to exposure to low oxygen tension have been determined. In addition, the implications of defective ATP release on human pathological conditions have been explored. This review provides a perspective on oxygen supply and the role that such a mechanism plays in meeting the oxygen needs of skeletal muscle. PMID:26336065

  2. Modelling the ATP production in mitochondria

    CERN Document Server

    Saa, Alberto

    2012-01-01

    We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We correct some inaccuracies in the BPLS original approximations and then analyze some of the dynamical properties of the model. We infer from exhaustive numerical explorations that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  3. Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

    Science.gov (United States)

    Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

    2017-04-01

    Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  4. Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

    2017-10-12

    The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

  5. Age-dependent impact of CaV3.2 T-type calcium channel deletion on myogenic tone and flow-mediated vasodilatation in small arteries

    DEFF Research Database (Denmark)

    Mikkelsen, Miriam F.; Björling, Karl; Jensen, Lars Jørn

    2016-01-01

    The myogenic response and flow-mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. CaV3.2 T-type calcium channels...... are expressed in both VSMCs and ECs of small arteries. The T-type channels are important drug targets but due to the lack of specific antagonists our understanding of the role of CaV3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow-mediated vasodilatation....... Our study shows important roles of the CaV3.2 T-type calcium channels in myogenic tone and flow-mediated vasodilation that disappear with aging. Since increased arterial tone is a risk factor for cardiovascular disease we conclude that CaV3.2 channels, by modulating pressure- and flow...

  6. Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection.

    Science.gov (United States)

    Liang, Yi-Dan; Bai, Wen-Jing; Li, Chen-Guang; Xu, Li-Hui; Wei, Hong-Xia; Pan, Hao; He, Xian-Hui; Ouyang, Dong-Yun

    2016-01-01

    Piperine is a phytochemical present in black pepper (Piper nigrum Linn) and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β) or high mobility group box-1 protein (HMGB1) release in LPS-primed bone marrow-derived macrophages and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK) activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine's inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis.

  7. Piperine suppresses pyroptosis and interleukin-1β release upon ATP triggering and bacterial infection

    Directory of Open Access Journals (Sweden)

    Yi-Dan Liang

    2016-10-01

    Full Text Available Piperine is a phytochemical present in black pepper (Piper nigrum Linn and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β or high mobility group box-1 protein (HMGB1 release in LPS-primed bone marrow-derived macrophages (BMDMs and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine’s inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis.

  8. Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

    Science.gov (United States)

    De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

    2014-01-01

    Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

  9. Mechanisms of vasodilatation in pregnancy: studies of the role of prostaglandins and nitric-oxide in changes of vascular reactivity in the in situ blood perfused mesentery of pregnant rats.

    OpenAIRE

    Chu, Z. M.; Beilin, L. J.

    1993-01-01

    1. To examine the possible mechanisms of the vasodilatation and blunted pressor responses in late pregnancy, we have studied vascular reactivity of the in situ blood perfused mesenteric resistance vessels of 18-20 day pregnant Wistar-Kyoto rats (WKY). 2. Intra-arterial mean blood pressure (MBP) was lower in pregnant rats than in nonpregnant controls. There was no significant difference in basal mesenteric perfusion pressure (BPP) between groups. 3. Vascular reactivity to electrical stimulatio...

  10. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. ME...

  11. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  12. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a dire...... shedding, possibly affecting the structural plasticity associated with learning and memory.......Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...

  13. Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

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    S.S. Frassetto

    2000-11-01

    Full Text Available The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation.

  14. Expression of ATP7B in normal human liver

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    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  15. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

    Science.gov (United States)

    Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

    2017-08-01

    Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Synergistic modulation of KCNQ1/KCNE1 K(+) channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP]i.

    Science.gov (United States)

    Kienitz, Marie-Cécile; Vladimirova, Dilyana

    2015-07-01

    Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion. Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. A Transient Rise in Free Mg2+Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

    2018-02-05

    For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  18. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

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    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  19. The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes.

    Science.gov (United States)

    Gasanov, S E; Kim, A A; Dagda, R K

    2016-07-01

    The effects of temperature and of the membrane-active protein CTII on the formation of nonbilayer structures in mitochondrial membranes were studied by (31)P-NMR. Increasing the temperature of isolated mitochondrial fractions correlated with an increase in ATP synthase activity and the formation of nonbilayer packed phospholipids with immobilized molecular mobility. Computer modeling was employed for analyzing the interaction of mitochondrial membrane phospholipids with the molecular surface of CTII, which behaves like a dicyclohexylcarbodiimide-binding protein (DCCD-BP) of the F0 group in a lipid phase. Overall, our studies suggest that proton permeability toroidal pores formed in mitochondrial membranes consist of immobilized nonbilayer-packed phospholipids formed via interactions with DCCD-BP. Our studies support the existence of a proton transport along a concentration gradient mediated via transit toroidal permeability pores which induce conformational changes necessary for mediating the catalytic activity of ATP synthase in the subunits of the F0-F1 complex.

  20. ATP released by injured neurons activates Schwann cells

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    Samuele eNegro

    2016-05-01

    Full Text Available Injured nerve terminals of neuromuscular junctions (NMJs can regenerate. This remarkable and complex response is governed by molecular signals that are exchanged among the cellular components of this synapse: motor axon nerve terminal (MAT, perisynaptic Schwann cells (PSCs, and muscle fibre. The nature of signals that govern MAT regeneration is ill-known. In the present study the spider toxin α-Latrotoxin has been used as tool to investigate the mechanisms underlying peripheral neuroregeneration. Indeed this neurotoxin induces an acute, specific, localized and fully reversible damage of the presynaptic nerve terminal, and its action mimics the cascade of events that leads to nerve terminal degeneration in injured patients and in many neurodegenerative conditions. Here we provide evidence of an early release by degenerating neurons of ATP as alarm messenger, that contributes to the activation of a series of intracellular pathways within SCs that are crucial for nerve regeneration: Ca2+, cAMP, ERK1/2, and CREB. These results contribute to define the cross-talk taking place among degenerating nerve terminals and PSCs, involved in the functional recovery of the NMJ.

  1. Assessment of flow-mediated vasodilatation (FMD) of the brachial artery: effects of technical aspects of the FMD measurement on the FMD response.

    Science.gov (United States)

    Bots, Michiel L; Westerink, Jan; Rabelink, Ton J; de Koning, Eelco J P

    2005-02-01

    The ability to assess endothelial function non-invasively with B-mode ultrasound has lead to its widespread application in a variety of studies. However, the absolute values obtained using this approach vary considerably across studies. We studied whether technical aspects of the methodology can explain the wide variety in absolute values across studies. A literature search was performed to identify published reports on flow-mediated vasodilatation (FMD) of the brachial artery published between 1992 and 2001. Information on type of equipment (wall track/B-mode), location of the measurement (antecubital fossa/upper arm), occlusion site (lower/upper arm), occlusion duration (min), and occlusion pressure was extracted. Patient characteristics were also extracted. For the healthy populations, mean FMD varied from 0.20 to 19.2%; for the coronary heart disease (CHD) patients FMD varied from -1.3 to 14%; for subjects with diabetes mellitus FMD varied from 0.75 to 12%. Compared with occlusion at the upper arm, lower arm occlusion was related to decreased FMD (mean difference in FMD -2.47%; 95% CI 0.55-4.39). An occlusion duration of > or =4.5 min was related to an increased FMD compared with an occlusion time of FMD differs widely between studies. There is a great overlap between populations (healthy, CHD, diabetics). Our findings suggest that the technical aspects of the measurements, the location, and the duration of the occlusion may explain some of these differences, whereas type of equipment, location of the measurement, and occlusion pressure do not.

  2. Downregulation of ATP-binding cassette subfamily C member 4 increases sensitivity to neoadjuvant radiotherapy for locally advanced rectal carcinoma.

    Science.gov (United States)

    Yu, Zhi-Qi; Zhang, Chang; Wang, Hao; Lao, Xin-Yuan; Chai, Rui; Gao, Xian-Hua; Cao, Guang-Wen; Fu, Chuan-Gang

    2013-05-01

    This study was designed to verify the effect of ATP-binding cassette subfamily C member 4 on radiosensitivity of locally advanced rectal carcinoma. The expression of ATP-binding cassette subfamily C member 4 protein in 121 pretreatment tissue samples from locally advanced rectal carcinoma patients was detected by immunohistochemistry. Pathological response to radiotherapy was evaluated according to tumor regression grading by postoperative histological examinations after they received long-course preoperative neoadjuvant radiotherapy, and the association between clinicopathological data and tumor regression grading was analyzed retrospectively. For further validation, short hairpin RNA was constructed and transfected into colorectal carcinoma cell line HT29. The knockdown efficiency was confirmed at both RNA and protein levels. The altered radiosensitivity was evaluated by methylthiazolyl tetrazolium assay, colony formation assay, flow cytometry, and Hoechst 33258 staining. Univariate analysis revealed that ATP-binding cassette subfamily C member 4 expression (p member 4 expression (p member 4 expression efficiently and persistently. Downregulation of ATP-binding cassette subfamily C member 4 expression significantly enhanced inhibition of cell proliferation, decreased colony formation capacity, and increased cell apoptosis induced by irradiation, as examined by a series of experiments in vitro. In addition, radiobiological parameters calculated according to the single-hit multitarget model were also decreased significantly. Our data indicate that ATP-binding cassette subfamily C member 4 may be a useful molecular marker in predicting radiosensitivity, and a potential target in improving the response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients.

  3. Distinct neurological disorders with ATP1A3 mutations

    Science.gov (United States)

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jóhannesson, Sigurður H.; Mikati, Mohamad A.; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J.; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J.; van den Maagdenberg, Arn; Vilsen, Bente

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  4. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Journey in guidelines for lipid management: From adult treatment panel (ATP-I to ATP-III and what to expect in ATP-IV

    Directory of Open Access Journals (Sweden)

    P G Talwalkar

    2013-01-01

    Full Text Available Adult Treatment Panel (ATP, an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual′s low density lipoprotein (LDL level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual′s overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist′s Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and

  6. Journey in guidelines for lipid management: From adult treatment panel (ATP)-I to ATP-III and what to expect in ATP-IV.

    Science.gov (United States)

    Talwalkar, P G; Sreenivas, C G; Gulati, Ashish; Baxi, Hemang

    2013-07-01

    Adult Treatment Panel (ATP), an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP) in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C) as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual's low density lipoprotein (LDL) level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual's overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist's Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and strong emphasis would

  7. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  8. ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.

    Science.gov (United States)

    Cai, Shi-Ying; Gautam, Samir; Nguyen, Trong; Soroka, Carol J; Rahner, Christoph; Boyer, James L

    2009-03-01

    Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane. ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs. ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase. ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

  9. Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

    Science.gov (United States)

    Schühle, Karola

    2012-01-01

    The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

  10. Effects of spermine NONOate and ATP on the thermal stability of hemoglobin

    Directory of Open Access Journals (Sweden)

    Bassam Rasha

    2012-08-01

    Full Text Available Abstract Background Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate, ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb. The effect of these molecules was examined by means of circular dichroism spectrometry (CD in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml was estimated via ellipticity change measurements at a heating rate of 1°C/min. Results Major results were: 1 spermine NONOate persistently decreased the hemoglobin unfolding temperature Tuirrespectively of the Na + /K + environment, 2 ATP instead increased the unfolding temperature by 3°C in both sodium-based and potassium-based buffers and 3 mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partial unfolding of alpha-helical structures even at room temperature. Conclusion The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulation of protein activity by small solutes in the cell.

  11. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

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    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  12. Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

    Science.gov (United States)

    Chène, Patrick; Rudloff, Joëlle; Schoepfer, Joseph; Furet, Pascal; Meier, Peter; Qian, Zhiyan; Schlaeppi, Jean-Marc; Schmitz, Rita; Radimerski, Thomas

    2009-01-07

    Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

  13. Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue

    Directory of Open Access Journals (Sweden)

    Schlaeppi Jean-Marc

    2009-01-01

    Full Text Available Abstract Background Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Results Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1 inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. Conclusion The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

  14. ATP1A3 Mutation in Adult Rapid-Onset Ataxia.

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    Kathleen J Sweadner

    Full Text Available A 21-year old male presented with ataxia and dysarthria that had appeared over a period of months. Exome sequencing identified a de novo missense variant in ATP1A3, the gene encoding the α3 subunit of Na,K-ATPase. Several lines of evidence suggest that the variant is causative. ATP1A3 mutations can cause rapid-onset dystonia-parkinsonism (RDP with a similar age and speed of onset, as well as severe diseases of infancy. The patient's ATP1A3 p.Gly316Ser mutation was validated in the laboratory by the impaired ability of the expressed protein to support the growth of cultured cells. In a crystal structure of Na,K-ATPase, the mutated amino acid was directly apposed to a different amino acid mutated in RDP. Clinical evaluation showed that the patient had many characteristics of RDP, however he had minimal fixed dystonia, a defining symptom of RDP. Successive magnetic resonance imaging (MRI revealed progressive cerebellar atrophy, explaining the ataxia. The absence of dystonia in the presence of other RDP symptoms corroborates other evidence that the cerebellum contributes importantly to dystonia pathophysiology. We discuss the possibility that a second de novo variant, in ubiquilin 4 (UBQLN4, a ubiquitin pathway component, contributed to the cerebellar neurodegenerative phenotype and differentiated the disease from other manifestations of ATP1A3 mutations. We also show that a homozygous variant in GPRIN1 (G protein-regulated inducer of neurite outgrowth 1 deletes a motif with multiple copies and is unlikely to be causative.

  15. Cervical anterior transpedicular screw fixation (ATPS)--Part II. Accuracy of manual insertion and pull-out strength of ATPS.

    Science.gov (United States)

    Koller, Heiko; Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H; Resch, Herbert; Hitzl, Wolfgang

    2008-04-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3-T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of "critical" pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm(2

  16. Thalidomide Improves the Intestinal Mucosal Injury and Suppresses Mesenteric Angiogenesis and Vasodilatation by Down-Regulating Inflammasomes-Related Cascades in Cirrhotic Rats.

    Science.gov (United States)

    Li, Tzu-Hao; Huang, Chia-Chang; Yang, Ying-Ying; Lee, Kuei-Chuan; Hsieh, Shie-Liang; Hsieh, Yun-Cheng; Alan, Lin; Lin, Han-Chieh; Lee, Shou-Dong; Tsai, Chang-Youh

    2016-01-01

    By blocking TNFα-related effects, thalidomide not only inhibits hepatic fibrogenesis but improves peripheral vasodilatation and portal hypertension in cirrhotic rats. Nonetheless, the investigation of thalidomide's effects on splanchnic and collateral microcirculation has been limited. Our study explored the roles of intestinal and mesenteric TNFα along with inflammasome-related pathway in relation to cirrhosis and the splanchnic/collateral microcirculation. Using in vivo and in vitro approaches, mechanisms of the effects of thalidomide on intestinal and mesenteric inflammatory, vasodilatory and angiogenic cascades-related abnormalities were explored in cirrhotic rats that had received 1-month thalidomide (C-T) treatment. In cirrhotic rats, high tumor necrosis factor (TNF)α, vascular endothelial growth factor (VEGF) and nitric oxide (NO)x levels were associated with the NOD-like receptors protein 3 (NLRP3), IL-1β and caspase-1 inflammasome over-expression in splenorenal shunt and mesenteric tissues. The thalidomide-related inhibition of mesenteric and splenorenal shunt inflammasome expression was accompanied by a significantly decreased intestinal mucosal injury and inflammasome immunohistochemical staining expression. Suppression of various angiogenic cascades, namely VEGF-NOS-NO, was paralleled by a decrease in mesenteric angiogenesis as detected by CD31 immunofluorescence staining and by reduced portosystemic shunting (PSS) in C-T rats. The down-regulation of the mesenteric and collateral vasodilatory VEGF-NOS-NO cascades resulted in a correction of vasoconstrictive hypo-responsiveness and in an attenuation of vasodilatory hyper-responsiveness when analyzed by in situ perfusion of the superior mesenteric arterial (SMA) and portosystemic collaterals. There was also a decrease in SMA blood flow and an increase in SMA resistance in the C-T rats. Additionally, acute incubation with thalidomide abolished TNFα-augmented VEGF-mediated migration of and tube

  17. ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

    Science.gov (United States)

    Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

    2015-07-03

    Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter*

    Science.gov (United States)

    Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

    2015-01-01

    Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [3H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. PMID:25991724

  19. ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes

    NARCIS (Netherlands)

    de Waart, Dirk R.; Naik, Jyoti; Utsunomiya, Karina S.; Duijst, Suzanne; Ho-Mok, Kam; Bolier, A. Ruth; Hiralall, Johan; Bull, Laura N.; Bosma, Piter J.; Oude Elferink, Ronald P. J.; Paulusma, Coen C.

    2016-01-01

    ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a

  20. Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology.

    Directory of Open Access Journals (Sweden)

    Sheng Li

    Full Text Available Periodontal disease (Periodontitis is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85% and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro

  1. Translocation of neuronal nitric oxide synthase to the plasma membrane by ATP is mediated by P2X and P2Y receptors

    Directory of Open Access Journals (Sweden)

    Ohnishi Takayuki

    2009-07-01

    Full Text Available Abstract Background The translocation of neuronal nitric oxide synthase (nNOS from the cytosol to the membrane is functionally coupled to the activation of N-methyl-D-aspartate (NMDA receptors at synapses. Whereas there is abundant evidence indicating that ATP and nitric oxide are involved in nociceptive transmission, whether nNOS is activated by ATP remains unknown. We recently established a fluorescence imaging system for examining nNOS translocation in PC12 cells expressing a yellow fluorescence protein-tagged nNOS N-terminal mutant, nNOSNT-YFP, and examined the effect of ATP on nNOS translocation using the system. Results The translocation of nNOS was induced by ATP in the presence of NMDA and forskolin, an adenylate cyclase activator. The purinergic P2X receptor agonist 2-MeSATP and the P2Y agonist UTP significantly enhanced nNOS translocation; and simultaneous stimulation with 2-MeSATP and UTP exhibited the same concentration-response curve for the translocation as obtained with ATP. ATP, 2-MeSATP, and UTP increased the intracellular Ca2+ concentration ([Ca2+]i in PC12 cells. Conversely, whereas the P2X receptor antagonist PPADS and the P2Y antagonist reactive blue-2 partially inhibited increases in the translocation of nNOS and [Ca2+]i by ATP, the non-selective P2 receptor antagonist suramin completely blocked them. In addition, the increase in the nNOS translocation by ATP was blocked by NMDA receptor antagonists and inhibitors of protein kinase A, protein kinase C, and Src kinase. Consistent with the expression of P2X and P2Y receptors in the spinal cord, ATP and UTP increased the [Ca2+]i in primary cultured spinal neurons. ATP potentiated and prolonged the [Ca2+]i increase produced by NMDA in the dorsal horn of the spinal cord. Furthermore, the selective P2X3/P2X2/3 antagonist A-317491 inhibited nNOS activation assessed by NO formation in spinal slices prepared from neuropathic pain model mice. Conclusion ATP is involved in n

  2. Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C.

    Science.gov (United States)

    Weng, Ju-Yun; Hsu, Tsan-Ting; Sun, Synthia H

    2008-05-15

    A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes. Copyright 2007 Wiley-Liss, Inc.

  3. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

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    Alexis Díaz-Vegas

    Full Text Available During exercise, skeletal muscle produces reactive oxygen species (ROS via NADPH oxidase (NOX2 while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

  4. Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-01-01

    Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved...... in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen...

  5. Phenomenological analysis of ATP dependence of motor protein

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  6. [ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

    Science.gov (United States)

    Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

    2002-01-01

    ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

  7. Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

    Directory of Open Access Journals (Sweden)

    Zhidan Xia

    2017-07-01

    Full Text Available Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC. However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1 protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

  8. ATP2A3 gene as an important player for resveratrol anticancer activity in breast cancer cells.

    Science.gov (United States)

    Izquierdo-Torres, Eduardo; Rodríguez, Gabriela; Meneses-Morales, Iván; Zarain-Herzberg, Angel

    2017-07-01

    The Ca2+ -ATPases from the Sarco/endoplasmic reticulum (SERCA) are fundamental for maintaining intracellular [Ca2+ ] homeostasis by pumping Ca2+ into the endoplasmic reticulum (ER) of eukaryotic cells. SERCA enzymes are encoded by three different genes (ATP2A1-3), whose expression occurs in a tissue and development stage-specific manner. It has been reported alterations in the expression of SERCA2 and SERCA3 pumps in different types of cancer: oral, lung, colon, stomach, central nervous system, thyroid, breast, and prostate. Resveratrol (RSV), a phytoalexin produced by a wide variety of plants in response to stress situations can modulate cellular processes involved in all stages of carcinogenesis. In this work, we used breast cancer cell lines (MCF-7 and MDA-MB-231) to evaluate mRNA levels of ATP2A2 and ATP2A3 genes in response to RSV treatment. Our results demonstrate that RSV treatment induced the expression of ATP2A3 gene in both cell lines in a time and concentration-dependent manner, while the expression of ATP2A2 gene remained unaffected. The RSV-induced expression of SERCA3 in these breast cancer cell lines produced decreased cell viability, triggered apoptosis and changes in cytosolic Ca2+ levels, as well as changes in the capacity for Ca2+ release by the ER. These data suggest an important participation of SERCA3 genes in RSV-mediated anti-tumor effect in breast cancer cell lines. Nevertheless, further research is needed to elucidate the molecular mechanisms underlying this effect. © 2017 Wiley Periodicals, Inc.

  9. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase.

    Science.gov (United States)

    Johnson, Larry; Atanasova, Kalina R; Bui, Phuong Q; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M

    2015-05-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the pro-inflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  10. Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis

    Science.gov (United States)

    Li, Yingniang; Wang, Jia; Li, Wenwen; Wang, Kai; Hong, Xiaoli; Zhao, Lu; Chen, Caiyong; Min, Junxia

    2017-01-01

    Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC. PMID:28692648

  11. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    Current standard methods for surveillance of microbial drinking water quality are culture based, which are laborious and time-consuming, where results not are available before one to three days after sampling. This means that the water may have been consumed before results on deteriorated water....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP......, microbial quality in distributed water, detection of aftergrowth, biofilm formation etc. This PhD project demonstrated that ATP levels are relatively low and fairly stable in drinking water without chlorine residual despite different sampling locations, different drinking water systems and time of year...

  12. ATP economy of force maintenance in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao

    2005-01-01

    PURPOSE: The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). METHODS: ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P......) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly during the 60-s...... contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P economy compared with those maintaining the force (3...

  13. Arabidopsis fructokinase-like protein associations are regulated by ATP.

    Science.gov (United States)

    Riggs, John W; Callis, Judy

    2017-05-10

    The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant form in vitro We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  14. Mechanism of ATP turnover inhibition in the EJC

    Science.gov (United States)

    Nielsen, Klaus H.; Chamieh, Hala; Andersen, Christian B.F.; Fredslund, Folmer; Hamborg, Kristiane; Le Hir, Hervé; Andersen, Gregers R.

    2009-01-01

    The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF3, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF3, suggesting that the use of ADP–AlF3 may be a valuable tool for examining DEAD-box ATPases in general. PMID:19033377

  15. Circular RNA Atp9b, a competing endogenous RNA, regulates the progression of osteoarthritis by targeting miR-138-5p.

    Science.gov (United States)

    Zhou, Zhi-Bin; Du, Di; Huang, Gao-Xiang; Chen, Aimin; Zhu, Lei

    2018-03-10

    Osteoarthritis (OA) is the most common joint disease and is mainly characterized by degradation of the articular cartilage. Recently, circular RNAs (circRNAs), novel noncoding RNAs with different biological functions and pathological implications, have been reported to be closely associated with various diseases. Growing evidence indicates that circRNAs act as competing endogenous RNAs (ceRNAs) that bind with microRNAs (miRNAs) and regulate their downstream functions. Here, we identified a new circRNA, circRNA_Atp9b, and further investigated its function in OA using a well-established mouse chondrocyte model. We demonstrated that circRNA_Atp9b expression was significantly up-regulated in mouse chondrocytes after stimulation with interleukin-1 beta (IL-1β), and that knockdown of circRNA_Atp9b promoted the expression of type II collagen while inhibiting the generation of MMP13, COX-2 and IL-6. Moreover, there was a negative correlation between the expression levels of circRNA_Atp9b and microRNA (miR)-138-5p, indicating that miR-138-5p also played a role in IL-1β-induced chondrocytes. Bioinformatics analysis predicted circRNA_Atp9b directly target miR-138-5p, which was validated by dual-luciferase assay. Further functional experiments revealed that down-regulation of miR-138-5p partly reversed the effects of circRNA_Atp9b on extracellular matrix (ECM) catabolism and inflammation. Taken together, these results suggest that circRNA_Atp9b regulates OA progression by modulating ECM catabolism and inflammation in chondrocytes via sponging miR-138-5p. Our findings provide novel insight into the regulatory mechanism of circRNA_Atp9b in OA and may contribute to establishing potential therapeutic strategies. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Theranostic Role of 32P-ATP as Radiopharmaceutical for the Induction of Massive Cell Death within Avascular Tumor Core.

    Science.gov (United States)

    Galiè, Mirco; Boschi, Federico; Scambi, Ilaria; Merigo, Flavia; Marzola, Pasquina; Altabella, Luisa; Lavagnolo, Umberto; Sbarbati, Andrea; Spinelli, Antonello E

    2017-01-01

    Drug inaccessibility to vast areas of the tumor parenchyma is amongst the major hurdles for conventional therapies. Treatment efficacy rapidly decreases with distance from vessels and most of the tumor cells survive therapy. Also, between subsequent cycles of treatment, spared cancer cells replace those killed near the vessels, improving their access to nutrients, boosting their proliferation rate, and thus enabling tumor repopulation. Because of their property of "acting at a distance," radioisotopes are believed to overcome the physical barrier of vascular inaccessibility. Methods A novel molecular imaging tool called Cerenkov Luminescence Imaging (CLI) was employed for the detection of Cerenkov radiation emitted by beta particles, allowing in vivo tracking of beta-emitters. More precisely we investigated using a xenograft model of colon carcinoma the potential use of 32P-ATP as a novel theranostic radiopharmaceutical for tracing tumor lesions while simultaneously hampering their growth. Results Our analyses demonstrated that 32P-ATP injected into tumor-bearing mice reaches tumor lesions and persists for days and weeks within the tumor parenchyma. Also, the high-penetrating beta particles of 32P-ATP exert a "cross-fire" effect that induces massive cell death throughout the entire tumor parenchyma including core regions. Conclusion Our findings suggest 32P-ATP treatment as a potential approach to complement conventional therapies that fail to reach the tumor core and to prevent tumor repopulation.

  17. CdTe amplification nanoplatforms capped with thioglycolic acid for electrochemical aptasensing of ultra-traces of ATP.

    Science.gov (United States)

    Shamsipur, Mojtaba; Farzin, Leila; Tabrizi, Mahmoud Amouzadeh; Shanehsaz, Maryam

    2016-12-01

    A "signal off" voltammetric aptasensor was developed for the sensitive and selective detection of ultra-low levels of adenosine triphosphate (ATP). For this purpose, a new strategy based on the principle of recognition-induced switching of aptamers from DNA/DNA duplex to DNA/target complex was designed using thioglycolic acid (TGA)-capped CdTe quantum dots (QDs) as the signal amplifying nano-platforms. Owing to the small size, high surface-to-volume ratio and good conductivity, quantum dots were immobilized on the electrode surface for signal amplification. In this work, methylene blue (MB) adsorbed to DNA was used as a sensitive redox reporter. The intensity of voltammetric signal of MB was found to decrease linearly upon ATP addition over a concentration range of 0.1nM to 1.6μM with a correlation coefficient of 0.9924. Under optimized conditions, the aptasensor was able to selectively detect ATP with a limit of detection of 45pM at 3σ. The results also demonstrated that the QDs-based amplification strategy could be feasible for ATP assay and presented a potential universal method for other small biomolecular aptasensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Physical activity opposes coronary vascular dysfunction induced during high fat feeding in mice.

    Science.gov (United States)

    Park, Yoonjung; Booth, Frank W; Lee, Sewon; Laye, Mathew J; Zhang, Cuihua

    2012-09-01

    The study's purpose was to investigate if physical activity initiated with the start of high-fat feeding would oppose development of endothelial dysfunction, and if it does, then to determine some potential mechanisms. C57BL/6 female mice were randomly divided into three groups: (1) control low-fat diet (LF-SED; 15% of calories from fat), (2) high-fat diet (HF-SED; 45% of calories from fat), and (3) HF diet given access to a voluntary running wheel (HF-RUN). Our hypothesis was that HF-RUN would differ in multiple markers of endothelial dysfunction from HF-SED after 10 weeks of 45%-fat diet, but would not differ from LF-SED. HF-RUN differed from HF-SED in nine determinations in which HF-SED either had decreases in (1) acetylcholine (ACh)-induced and flow-induced vasodilatations in isolated, pressurized coronary arterioles, (2) heart phosphorylated endothelial nitric oxide synthase (p-eNOS/eNOS) protein, (3) coronary arteriole leptin (ob) receptor protein, (4) phosphorylated signal transducer and activator of transcription 3 (p-STAT3/STAT3) protein, and (5) coronary arteriole superoxide dismutase 1 protein; or had increases in (6) percentage body fat, (7) serum leptin, (8) coronary arteriole suppressor of cytokine signalling 3 (SOCS3) protein, and (9) coronary arteriole gp91(phox) protein. Higher endothelium-dependent vasodilatation by ACh or leptin was abolished with incubation of NOS inhibitor N(G)-nitro-l-arginine-methyl ester (l-NAME) in LF-SED and HF-RUN groups. Further, impaired ACh-induced vasodilatation in HF-SED was normalized by apocynin or TEMPOL to LF-SED and HF-RUN. These findings demonstrate multiple mechanisms (eNOS, leptin and redox balance) by which voluntary running opposes the development of impaired coronary arteriolar vasodilatation during simultaneous high-fat feeding.

  19. Effect of ATP on intracellular pH in pancreatic ducts involves P2X7 receptors

    DEFF Research Database (Denmark)

    Henriksen, Katerine L; Novak, Ivana

    2003-01-01

    which P2 receptors might be involved. Ducts were obtained from rat pancreas, and the pH sensitive fluorophore BCECF was used to measure pHi and recovery rates from cellular acidosis induced by ammonium pre-pulses. In order to reveal Na+/H+ exchange, Cl-/HCO3- exchange or a Na+-HCO3- cotransport...... unmasked by cellular acidosis. Presumably, ATP alone does not stimulate HCO3- secretion in pancreatic ducts....

  20. Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation

    Directory of Open Access Journals (Sweden)

    Michael Bording-Jorgensen

    2017-01-01

    Full Text Available Background: Nod-like receptor family, pyrin domain containing 3 (NLRP3 is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome that triggers the maturation and secretion of interleukin (IL-1β and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. Methods: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1β; cytokines and reactive oxygen species (ROS were measured by ELISA and DCFDA, respectively. Results: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. Conclusion: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

  1. Abnormal expression of ATP1A1 and ATP1A2 in breast cancer [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Alexey Bogdanov

    2017-01-01

    Full Text Available Breast cancer is the first in incidence and the second in death among all solid tumors occurring in women. The identification of molecular genetic abnormalities in breast cancer is important to improve the results of treatment. In the present study, we analyzed microarray data of breast cancer expression profiling (NCBI GEO database, accession GSE65194, focusing on Na+/K+-ATPase coding genes. We found overexpression of the ATP1A1 and down-regulation of the ATP1A2. We expect that our research could help to improve the understanding of predictive and prognostic features of breast cancer.

  2. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    Science.gov (United States)

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  3. Prevention of phosphine-induced cytotoxicity by nutrients in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Marzieh Rashedinia

    2016-01-01

    Interpretation & conclusions: The results supported the hypothesis that phosphine-induced cytotoxicity was due to decrease of ATP levels. ATP suppliers could prevent its toxicity by generating ATP through glycolysis. α-keto compounds such as dihydroxyacetone and α-ketoglutarate may bind to phosphine and restore mitochondrial respiration.

  4. Bioluminometric assay of ATP in mouse brain: Determinant factors ...

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  5. Electrochemical Investigation of the Interaction between Catecholamines and ATP.

    Science.gov (United States)

    Taleat, Zahra; Estévez-Herrera, Judith; Machado, José D; Dunevall, Johan; Ewing, Andrew G; Borges, Ricardo

    2018-02-06

    The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

  6. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...

  7. Diversity and regulation of ATP sulfurylase in photosynthetic organisms

    Directory of Open Access Journals (Sweden)

    Laura ePrioretti

    2014-11-01

    Full Text Available ATP sulfurylase (ATPS catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper challenge this view and suggest that ATPSes may have a crucial regulatory role in sulfate assimilation, at least in algae.In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s expression. Special attention is given to algae ATPSes. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPSes revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. For instance, algae ATPSes show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. It is interesting that, at least with respect to the number of cysteines, the ATPSes of eukaryotic algae are closer to the marine cyanobacteria of the genera Synechococcus and Prochlorococcus and are more distant from freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater.

  8. Familial Hemiplegic Migraine With ATP1A2 Mutations

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2007-05-01

    Full Text Available Three children with prolonged hemiplegia following severe unilateral headache and having mutations in ATP1A2 are reported from UCLA School of Medicine, Los Angeles, CA; University Children’s Hospital, Zurich, Switzerland; and Wake Forest University School of Medicine, Winston-Salem, NC.

  9. Distinct neurological disorders with ATP1A3 mutations

    NARCIS (Netherlands)

    Heinzen, E.L.; Arzimanoglou, A.; Brashear, A.; Clapcote, S.J.; Gurrieri, F.; Goldstein, D.B; Johannesson, S.H.; Mikati, M.A.; Neville, B.; Nicole, S.; Ozelius, L.J.; Poulsen, H.; Schyns, T.; Sweadner, K.J.; Maagdenberg, A. van den; Vilsen, B.; Koenderink, J.B.

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the alpha3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological

  10. Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

    Science.gov (United States)

    Kolesnikov, Michael P.; Telegina, Taisiya A.; Lyudnikova, Tamara A.; Kritsky, Mikhail S.

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10 20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer left( {{text{FlH}}^ bullet } right) and ADP are involved.

  11. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  12. Hair bundles are specialized for ATP delivery via creatine kinase.

    NARCIS (Netherlands)

    Shin, J.B.; Streijger, F.; Beynon, A.J.; Peters, T.; Gadzala, L.; McMillen, D.; Bystrom, C.; Zee, C.E.E.M. van der; Wallimann, T.; Gillespie, P.G.

    2007-01-01

    When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to

  13. Treatment of heterotopic ossification through remote ATP hydrolysis.

    Science.gov (United States)

    Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

    2014-09-24

    Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. Copyright © 2014, American Association for the Advancement of Science.

  14. Clipboard: New paradigm for ATP synthesis and consumption

    Indian Academy of Sciences (India)

    2011-03-14

    Mar 14, 2011 ... Home; Journals; Journal of Biosciences; Volume 36; Issue 1. Clipboard: New paradigm for ATP synthesis and consumption. C Channakeshava. Volume 36 Issue 1 March 2011 pp 3-4. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/jbsc/036/01/0003-0004 ...

  15. Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.

    Science.gov (United States)

    Li, Jian-Hu; Wang, Yi-Fu; Ha, Wei; Liu, Yan; Ding, Li-Sheng; Li, Bang-Jing; Zhang, Sheng

    2013-09-09

    A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of α-cyclodextrins/chitosan-graft-poly(ethylene glycol) methacrylate. Upon entrapped GOD into these capsules, the addition of glucose could trigger proton-motive force and then drive the rotation of ATPase to synthesize ATP.

  16. Activated sludge optimization using ATP in pulp and paper industry.

    Science.gov (United States)

    Bäckman, Göran; Gytel, Ulla

    2015-01-01

    The activated sludge process is an old technology, but still the most commonly used one for treatment of wastewater. Despite the wide spread usage the technology still suffers from instability (Tandoi et al. 2006) and high operating cost. Activated sludge processes often carry a large solids inventory. Managing the total inventory without interference is the key component of the optimization process described in this paper. Use of nutrients is common in pulp and paper effluent treatment. Feeding enough nutrients to support the biomass growth is a delicate balance. Overfeeding or underfeeding of nutrients can result in higher costs. Detrimental substances and toxic components in effluents entering a biological treatment system can cause severe, long lasting disturbances (Hynninen & Ingman 1998; Bergeron & Pelletier 2004). A LumiKem test kit is used to measure biological activity with adenosine triphosphate (ATP) in a pulp and paper mill. ATP data are integrated with other standardized mill parameters. Measurements of active volatile suspended solids based on ATP can be used to quantify the living biomass in the activated sludge process and to ensure that sufficient biomass is present in order to degrade the wastewater constituents entering the process. Information about active biomass will assist in optimizing sludge inventories and feeding of nutrients allowing the living biomass to re-populate to create optimal efficiency. ATP measurements can also be used to alert operators if any components toxic to bacteria are present in wastewater. The bio stress index represents the stress level experienced by the microbiological population. This parameter is very useful in monitoring toxicity in and around bioreactors. Results from the wastewater process optimization and ATP measurements showed that treatment cost could be reduced by approximately 20-30% with fewer disturbances and sustained biological activity compared to the reference period. This was mainly achieved by

  17. Effect of ATP-binding cassette subfamily B member 1 on bovine blastocyst implantation.

    Science.gov (United States)

    Mori, M; Kuwano, T; Kamori, T; Isozaki, Y; Nishihara, T; Yamauchi, N; Hattori, M-A

    2014-03-15

    The ATP-binding cassette subfamily B member 1 (ABCB1) is an efflux transporter that excretes xenobiotics and waste matter. High expression of ABCB1 induced by forskolin (FSK) and rifampicin (RIF) in the bovine blastocysts reportedly improves the cellular quality. In the present study, interferon-α, similar to FSK and RIF, was highly potent in inducing the expression of ABCB1 in the bovine blastocysts but did not exhibit an additive effect with FSK and RIF. Bovine blastocysts stimulated by the combined treatment with FSK, RIF, and interferon-α to express high levels of ABCB1 displayed better freezing resistance as indicated by higher cell numbers in post thawing cultures. On transfer to recipients, such embryos established pregnancies with significantly higher frequencies in repeat breeder cows rather than normal ones. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    Science.gov (United States)

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  19. Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells.

    Science.gov (United States)

    Glass-Marmor, L; Morgenstern, H; Beitner, R

    1996-10-17

    Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of four different calmodulin antagonists: thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), CGS 9343B (1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a] [4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2 H-benzimidazol-2-one (1:1) maleate), clotrimazole (1-(alpha-2-chlorotrityl)imidazole) and bifonazole (1-(alpha-biphenyl-4-ylbenzyl)imidazole), on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in B16 melanoma cells. We found that all four substances significantly reduced the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP, in a dose- and time-dependent manner. Cell viability was reduced in a close correlation with the fall in ATP. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate did not result from the cytotoxic effects of the calmodulin antagonists, since their content was already reduced before any cytotoxic effect was observed. These findings suggest that the fall in the levels of the two signal molecules of glycolysis, induced by the calmodulin antagonists, causes a reduction in glycolysis and ATP levels, which eventually leads to cell death. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, these substances are most promising agents in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cell growth.

  20. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  1. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  2. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph

    2017-01-01

    Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened......G or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin......, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards...

  3. Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

    CSIR Research Space (South Africa)

    Salisu, S

    2011-05-01

    Full Text Available In research directed at the development of adenine triphosphate (ATP) analogs as potential glutamine synthetase (GS) inhibitors, adenine and allopurinol derivatives have been synthesized either as novel ATP analogs or as scaffolds...

  4. A functional 12T-insertion polymorphism in the ATP1A1 promoter confers decreased susceptibility to hypertension in a male Sardinian population.

    Directory of Open Access Journals (Sweden)

    Victoria L Herrera

    -reabsorption in the kidney thereby decreasing sodium-associated risk for hypertension and sodium-induced endothelial stiffness and dysfunction. Altogether, data support ATP1A1 as a hypertension susceptibility gene in a male Sardinian population, and mandate further investigation of its involvement in hypertension in the general population.

  5. The metabolic inhibitor antimycin A can disrupt cell-to-cell communication by an ATP- and Ca(2+)-independent mechanism.

    Science.gov (United States)

    Plaisance, Isabelle; Duthe, Fabien; Sarrouilhe, Denis; Hervé, Jean-Claude

    2003-11-01

    In cardiac myocytes of new-born rats, the degree of intercellular communication through gap junctional channels closely depends on the metabolic state of the cells. In contrast, in stably transfected HeLa cells expressing rat cardiac connexin43 (Cx43, the main channel-forming protein present in ventricular myocytes), a major part of junctional communication persisted in ATP-depleted conditions, in the presence of a metabolic inhibitor (KCN) or of a broad spectrum inhibitor of protein kinases (H7). However, another metabolic inhibitor, antimycin A, which like cyanide inhibits electron transfer in the respiratory chain, totally interrupted cell-to-cell communication between Cx43-HeLa cells, even in whole-cell conditions, when ATP (5 mM) was present. Antimycin A caused a modest increase in cytosolic calcium concentration; however, junctional uncoupling still occurred when this rise was prevented. Conditions of ischemic insult (e.g. ischemia or chemical hypoxia) frequently cause the activation of protein kinases, particularly of Src and MAP kinases, and such activations are known to markedly disrupt gap junctional communication. Antimycin-induced junctional uncoupling occurred even in the presence of inhibitors of these kinases. Antimycin A appears able to cause junctional uncoupling either through the ATP depletion it induces as a metabolic poison or via a direct action on gap junction constituents.

  6. Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

    OpenAIRE

    Tomomi Ando; Hiromi Imamura; Ryosuke Suzuki; Hideki Aizaki; Toshiki Watanabe; Takaji Wakita; Tetsuro Suzuki

    2012-01-01

    Adenosine 5'-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energi...

  7. The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2011-01-01

    selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects...... or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological...... compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis....

  8. Arsenic Alters ATP-Dependent Ca2+ Signaling in Human Airway Epithelial Cell Wound Response

    Science.gov (United States)

    Sherwood, Cara L.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2011-01-01

    Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca2+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., arsenic reduces purinergic Ca2+ signaling in a dose-dependent manner and results in a reshaping of the Ca2+ signaling response to localized wounds. We next examined arsenic effects on two purinergic receptor types: the metabotropic P2Y and ionotropic P2X receptors. Arsenic inhibited both P2Y- and P2X-mediated Ca2+ signaling responses to ATP. Both inhaled and ingested arsenic can rapidly reach the airway epithelium where purinergic signaling is essential in innate immune functions (e.g., ciliary beat, salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease. PMID:21357385

  9. Creatine kinase-mediated ATP supply fuels actin-based events in phagocytosis.

    Directory of Open Access Journals (Sweden)

    Jan W P Kuiper

    2008-03-01

    Full Text Available Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B, an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

  10. Discovery and biochemical characterization of selective ATP competitive inhibitors of the human mitotic kinesin KSP.

    Science.gov (United States)

    Rickert, Keith W; Schaber, Michael; Torrent, Maricel; Neilson, Lou Anne; Tasber, Edward S; Garbaccio, Robert; Coleman, Paul J; Harvey, Diane; Zhang, Yun; Yang, Yi; Marshall, Gary; Lee, Ling; Walsh, Eileen S; Hamilton, Kelly; Buser, Carolyn A

    2008-01-15

    The kinesin spindle protein (KSP, also known as Eg5) is essential for the proper separation of spindle poles during mitosis, and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature. However, most appear to share a common induced-fit allosteric binding site, suggesting a common mechanism of inhibition. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior, consistent with binding within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP-utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.

  11. The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling.

    Science.gov (United States)

    Civril, Filiz; Bennett, Matthew; Moldt, Manuela; Deimling, Tobias; Witte, Gregor; Schiesser, Stefan; Carell, Thomas; Hopfner, Karl-Peter

    2011-10-28

    RIG-I detects cytosolic viral dsRNA with 5' triphosphates (5'-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5'-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.

  12. Glycolysis, but not Mitochondria, responsible for intracellular ATP distribution in cortical area of podocytes.

    Science.gov (United States)

    Ozawa, Shota; Ueda, Shuko; Imamura, Hiromi; Mori, Kiyoshi; Asanuma, Katsuhiko; Yanagita, Motoko; Nakagawa, Takahiko

    2015-12-18

    Differentiated podocytes, a type of renal glomerular cells, require substantial levels of energy to maintain glomerular physiology. Mitochondria and glycolysis are two major producers of ATP, but the precise roles of each in podocytes remain unknown. This study evaluated the roles of mitochondria and glycolysis in differentiated and differentiating podocytes. Mitochondria in differentiated podocytes are located in the central part of cell body while blocking mitochondria had minor effects on cell shape and migratory ability. In contrast, blocking glycolysis significantly reduced the formation of lamellipodia, a cortical area of these cells, decreased the cell migratory ability and induced the apoptosis. Consistently, the local ATP production in lamellipodia was predominantly regulated by glycolysis. In turn, synaptopodin expression was ameliorated by blocking either mitochondrial respiration or glycolysis. Similar to differentiated podocytes, the differentiating podocytes utilized the glycolysis for regulating apoptosis and lamellipodia formation while synaptopodin expression was likely involved in both mitochondrial OXPHOS and glycolysis. Finally, adult mouse podocytes have most of mitochondria predominantly in the center of the cytosol whereas phosphofructokinase, a rate limiting enzyme for glycolysis, was expressed in foot processes. These data suggest that mitochondria and glycolysis play parallel but distinct roles in differentiated and differentiating podocytes.

  13. RNA Remodeling Activity of DEAD Box Proteins Tuned by Protein Concentration, RNA Length, and ATP.

    Science.gov (United States)

    Kim, Younghoon; Myong, Sua

    2016-09-01

    DEAD box RNA helicases play central roles in RNP biogenesis. We reported earlier that LAF-1, a DEAD box RNA helicase in C. elegans, dynamically interacts with RNA and that the interaction likely contributes to the fluidity of RNP droplets. Here we investigate the molecular basis of the interaction of RNA with LAF-1 and its human homolog, DDX3X. We show that both LAF-1 and DDX3X, at low concentrations, are monomers that induce tight compaction of single-stranded RNA. At high concentrations, the proteins are multimeric and dynamically interact with RNA in an RNA length-dependent manner. The dynamic LAF-1-RNA interaction stimulates RNA annealing activity. ATP adversely affects the RNA remodeling ability of LAF-1 by suppressing the affinity, dynamics, and annealing activity of LAF-1, suggesting that ATP may promote disassembly of the RNP complex. Based on our results, we postulate a plausible molecular mechanism underlying the dynamic equilibrium of the LAF-1 RNP complex. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    NARCIS (Netherlands)

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C. D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    2014-01-01

    Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we

  15. A comparative study of ATPase subunit 9 (Atp9) gene between ...

    African Journals Online (AJOL)

    ATPase subunit 9 gene (Atp9) is an important functional gene in mitochondria, and is closely related with energy supply. RNA editing of atp9 gene was associated with male sterility in plants. In this study, the atp9 gene in soybeans was cloned from a soybean cytoplasmic male sterile line NJCMS2A and its maintainer line ...

  16. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2015-01-01

    effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP...

  17. A rapid and convenient method for preparing salt-free (. gamma. -/sup 32/P)ATP

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, J.L.; Avruch, J.

    1981-09-15

    (..gamma..-/sup 32/P)ATP is prepared by an existing enzymatic method that yields approximately 95% incorporation of /sup 32/P into ATP. A rapid and convenient method for purifying the (..gamma..-/sup 32/P)ATP which results in a product free of both salt and buffer is reported.

  18. Glycolysis and ATP degradation in cod ( Gadus morhua ) at subzero temperatures in relation to thaw rigor

    DEFF Research Database (Denmark)

    Cappeln, Gertrud; Jessen, Flemming

    2001-01-01

    Glycolysis was shown to occur during freezing of cod of decrease in glycogen and an increase in lactate. In addition, the ATP content decreased during freezing. Synthesis of ATP was measured as degradation of glycogen. During storage at -9 and - 12 degreesC it was found that degradation of ATP...

  19. Ionotropic ATP receptors in neuronal-glial communication.

    Science.gov (United States)

    Lalo, Ulyana; Verkhratsky, Alexei; Pankratov, Yuri

    2011-04-01

    In the central nervous system ATP is released from both neurones and astroglial cells acting as a homo- and heterocellular neurotransmitter. Glial cells express numerous purinoceptors of both ionotropic (P2X) and metabotropic (P2Y) varieties. Astroglial P2X receptors can be activated by ongoing synaptic transmission and can mediate fast local signalling through elevation in cytoplasmic Ca(2+) and Na(+) concentrations. These ionic signals can be translated into various physiological messages by numerous pathways, including release of gliotransmitters, metabolic support of neurones and regulation of activity of postsynaptic glutamate and GABA receptors. Ionotropic purinoceptors represent a novel pathway of glia-driven modulation of synaptic signalling that involves the release of ATP from neurones and astrocytes followed by activation of P2X receptors which can regulate synaptic activity by variety of mechanisms expressed in both neuronal and glial compartments. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Students' Interdisciplinary Reasoning about "High-Energy Bonds" and ATP

    CERN Document Server

    Dreyfus, Benjamin W; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F

    2012-01-01

    Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectiv...

  1. Students' interdisciplinary reasoning about "high-energy bonds" and ATP

    Science.gov (United States)

    Dreyfus, Benjamin W.; Geller, Benjamin D.; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F.

    2013-01-01

    Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectives.

  2. Motor pathway excitability in ATP13A2 mutation carriers

    DEFF Research Database (Denmark)

    Zittel, S; Kroeger, J; van der Vegt, J P M

    2012-01-01

    OBJECTIVE: To describe excitability of motor pathways in Kufor-Rakeb syndrome (PARK9), an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration caused by a mutation in the ATP13A2 gene, using transcranial magnetic stimulation (TMS). METHODS: Five members of a Chilean family...... with an ATP13A2 mutation (one affected mutation carrier (MC) with a compound heterozygous mutation, 4 asymptomatic MC with a single heterozygous mutation) and 11 healthy subjects without mutations were studied. We measured motor evoked potentials (MEP), the contralateral silent period (cSP), short interval...... intracortical inhibition (SICI), intracortical facilitation (ICF), short latency afferent inhibition (SAI) as markers of intracortical intrahemispheric inhibition/facilitation and the ipsilateral silent period (iSP) and paired-pulse interhemispheric inhibition (IHI) to probe interhemispheric motor interactions...

  3. Kinetics of signaling-DNA-aptamer-ATP binding

    Science.gov (United States)

    Nakamura, Issei; Shi, An-Chang; Nutiu, Razvan; Yu, Jasmine M. Y.; Li, Yingfu

    2009-03-01

    DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at 23°C . A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to a simple procedure to deduce relevant kinetic reactions and their rate constants. A comparison between theory and experiments indicates that the previously established bimolecular DNA-ATP binding does not provide a complete description of the experimental data. Side reactions such as trimolecular complexation are proposed. Rate constants of the model are determined by comparing the model predictions and experiments. Good agreements between the model and experiments have been obtained. Possible blocking reactions by the misfolded DNA aptamer are also discussed.

  4. Structure and mechanism of ATP-dependent phospholipid transporters

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Poulsen, Lisbeth Rosager; Bailly, Aurélien

    2015-01-01

    , despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic......Background ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review This review aims to identify common mechanistic features...... in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit. Further...

  5. Contributions of ADP and ATP to the increase in skeletal muscle blood flow after manual acupuncture stimulation in rats.

    Science.gov (United States)

    Nagaoka, S; Shinbara, H; Okubo, M; Kawakita, T; Hino, K; Sumiya, E

    2016-06-01

    To investigate the contributions of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to the increase in skeletal muscle blood flow (MBF) observed following manual acupuncture (MA) stimulation in rats. Male Sprague-Dawley rats were used as experimental animals (300-370 g, n=40). MA was applied to the right tibialis anterior muscle (TA) for 1 min using a stainless steel acupuncture needle. In eight rats, high-performance liquid chromatography with the microdialysis technique was used to measure local extracellular concentrations of ATP, ADP, adenosine monophosphate (AMP), and adenosine in the TA. In the remaining 32 rats, fluorescent microspheres (15 µm in diameter) were used to measure MBF in the TA following pre-treatment with either the P2 receptor antagonist suramin (100 mg/kg intra-arterially) or saline (control) (n=16 each). Rats receiving MA (Suramin+MA and Saline+MA groups, n=8 each) were compared with untreated rats (Suramin and Saline groups, n=8). MA significantly increased the local extracellular concentration of ATP, ADP, and adenosine (pvs 30 min after MA). In addition, MA significantly increased MBF in rats pre-treated with saline or suramin (pvs Saline+MA; pvs Suramin+MA, respectively). However, suramin significantly suppressed this MA-induced increase in MBF (pvs Suramin+MA). These results suggest that both ATP and ADP partially contribute to the MA-induced increase in MBF via P2 receptors. However, further studies are needed to clarify the contributions of other vasodilators. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  6. Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer.

    Science.gov (United States)

    Li, Yapiao; Sun, Linlin; Zhao, Qiang

    2017-11-01

    We developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl 2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5μM to 1mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

    Science.gov (United States)

    Nath, Sunil

    2016-12-01

    As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Alternative mitochondrial functions in cell physiopathology: beyond ATP production

    Directory of Open Access Journals (Sweden)

    Kowaltowski A.J.

    2000-01-01

    Full Text Available It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

  9. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

    Full Text Available Abstract There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx or out (efflux of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]. ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders.

  10. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  11. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes.

    Science.gov (United States)

    Hurst, S E; Minkin, S C; Biggerstaff, J; Dhar, M S

    2012-01-01

    Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  12. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    S. E. Hurst

    2012-01-01

    Full Text Available Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs, p38, and p44/42. Additionally, glucose transporter-1 (GLUT1 was significantly upregulated; no changes in glucose transporter-4 (GLUT4 expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  13. atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

    Science.gov (United States)

    2013-01-01

    Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

  14. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank B; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout......-perfused coronary circulation, [ATP] increased as the perfusate moved through the vessels in the presence of ARL 67156. When ATP was added to the inflowing saline, most ATP disappeared during passage of the coronary bed when ARL 67156 was absent but not when it was present. We conclude that rainbow trout...

  15. Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation

    Science.gov (United States)

    Matsukawa, Toshihiro; Izawa, Kumi; Isobe, Masamichi; Takahashi, Mariko; Maehara, Akie; Yamanishi, Yoshinori; Kaitani, Ayako; Okumura, Ko; Teshima, Takanori; Kitamura, Toshio; Kitaura, Jiro

    2016-01-01

    Objective Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide–LMIR3 interaction in the development of IBD. Design The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3−/−, mast cell-deficient KitW-sh/W-sh, KitW-sh/W-shLMIR3−/− or KitW-sh/W-sh mice engrafted with WT or LMIR3−/− bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. Results LMIR3 deficiency exacerbated DSS-induced colitis in mice. KitW-sh/W-sh mice harbouring LMIR3−/− mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide–LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide–LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. Conclusions LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3−/− mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide–LMIR3 binding. PMID:25673319

  16. Evaluation of potassium ion as the endothelium-derived hyperpolarizing factor (EDHF) in the bovine coronary artery.

    Science.gov (United States)

    Nelli, Silvia; Wilson, William S; Laidlaw, Hilary; Llano, Andrea; Middleton, Susan; Price, Andrew G; Martin, William

    2003-07-01

    1. This study explored the role of the potassium ion in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine coronary artery. 2. Bradykinin-induced, EDHF-mediated vasodilatation was blocked by the Na(+)-K(+) ATPase inhibitor, ouabain (1 micro M), in a time-dependent manner, with maximal blockade seen after 90 min. In contrast, the K(IR) channel inhibitor, Ba(2+) (30 micro M), had no effect. 3. When the potassium content of the bathing solution was increased in a single step from 5.9 to 7-19 mM, powerful vasodilatation (max. 75.9+/-3.6%) was observed. Vasodilatation was transient and, consequently, cumulative addition of potassium produced little vasodilatation, with vasoconstriction predominating at the higher concentrations. 4. The magnitude of potassium-induced vasodilatation was similar in endothelium-containing and endothelium-denuded rings, and was unaffected by Ba(2+) (30 micro M), but abolished by ouabain (1 micro M). 5. Ouabain (1 micro M, 90 min) powerfully blocked bradykinin-induced, nitric oxide-mediated vasodilatation as well as that induced by the nitrovasodilator, glyceryl trinitrate, but that induced by the K(ATP) channel opener, levcromakalim, was hardly affected. 6. Thus, activation of Na(+)-K(+) ATPase is likely to be involved in the vasodilator responses of the bovine coronary artery to both nitric oxide and EDHF. These findings, together with the ability of potassium to induce powerful, ouabain- but not Ba(2+)-sensitive, endothelium-independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.

  17. Pharmacological and molecular comparison of K(ATP) channels in rat basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Edvinsson, Lars; Olesen, Jes

    2006-01-01

    ATP-sensitive potassium (K(ATP)) channels play an important role in the regulation of cerebral vascular tone. In vitro studies using synthetic K(ATP) channel openers suggest that the pharmacological profiles differ between rat basilar arteries and rat middle cerebral arteries. To address this issue......, we studied the possible involvement of endothelial K(ATP) channels by pressurized arteriography after luminal administration of synthetic K(ATP) channel openers to rat basilar and middle cerebral arteries. Furthermore, we examined the mRNA and protein expression profile of K(ATP) channels to rat...... basilar and middle cerebral arteries using quantitative real-time PCR (Polymerase Chain Reaction) and Western blotting, respectively. In the perfusion system, we found no significant responses after luminal application of three K(ATP) channel openers to rat basilar and middle cerebral arteries...

  18. ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jorgensen, N R; Geist, S T; Civitelli, R

    1997-01-01

    stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...... mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein...... connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U...

  19. A metabolic link between mitochondrial ATP synthesis and liver glycogen metabolism: NMR study in rats re-fed with butyrate and/or glucose

    Directory of Open Access Journals (Sweden)

    Beauvieux Marie-Christine

    2011-06-01

    phosphorylation yield and to induce a glucose-sparing effect, delayed the transient increase in mitochondrial ATP turnover and hence energy contribution to glycogen metabolism.

  20. Divergent action of calcium channel blockers on ATP-binding cassette protein expression.

    Science.gov (United States)

    Hasegawa, Kazuhiro; Wakino, Shu; Kanda, Takeshi; Yoshioka, Kyoko; Tatematsu, Satoru; Homma, Koichiro; Takamatsu, Ichiro; Sugano, Naoki; Hayashi, Koichi

    2005-12-01

    Calcium channel blockers (CCBs) are widely used in clinical practice, and have been reported to be effective in preventing the progression of atherosclerosis. We examined whether various types of calcium channel blockers affected the expression of ATP binding cassette transporter A1 (ABCA1), a factor contributing to anti-atherogenesis. Undifferentiated monocytic cell line, THP-1 cells were maintained in RPMI 1640 medium and treated with different kinds of calcium channel blockers. Among the calcium channel blockers tested, aranidipine and efonidipine increased ABCA1 protein expression without an increase in ABCA1 mRNA expression, whereas other calcium channel blockers (eg, nifedipine, amlodipine, and nicardipine) or T-type calcium channel blockers (eg, mibefradil and nickel chloride) failed to upregulate ABCA1 expression. H89, a protein kinase A inhibitor inhibited the aranidipine-induced ABCA1 protein expression, whereas genistein (a tyrosine kinase inhibitor), or AG490 (a JAK-2 inhibitor) had no effects. Neither of these inhibitors suppressed the efonidipine-induced ABCA1 protein expression. Intracellular cAMP levels were elevated only by aranidipine, but not by efonidipine. In conclusion, aranidipine and efonidipine have the ability to induce ABCA1 protein by distinct mechanisms; protein kinase A is involved in the aranidipine-induced ABCA1 upregulation. This non-class effect of calcium channel blockers may potentially offer beneficial action in the treatment of hypertensive subjects with atherosclerosis.

  1. Acute simvastatin inhibits K ATP channels of porcine coronary artery myocytes.

    Directory of Open Access Journals (Sweden)

    Sai Wang Seto

    Full Text Available BACKGROUND: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular KATP channel gatings are unknown. METHODS: Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca(2+]i, [ATP]i and [glucose]o uptake measurements. RESULTS: The cromakalim (10 nM to 10 µM- and pinacidil (10 nM to 10 µM-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM. Simvastatin (1, 3 and 10 µM suppressed (in okadaic acid (10 nM-sensitive manner cromakalim (10 µM- and pinacidil (10 µM-mediated opening of whole-cell KATP channels of arterial myocytes. Simvastatin (10 µM and AICAR (1 mM elicited a time-dependent, compound C (1 µM-sensitive [(3H]-2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2-30 min- and concentration (0.1-10 µM-dependent increase by simvastatin of p-AMPKα-Thr(172 and p-PP2A-Tyr(307 expression was observed. The enhanced p-AMPKα-Thr(172 expression was inhibited by compound C, ryanodine (100 µM and KN93 (10 µM. Simvastatin-induced p-PP2A-Tyr(307 expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM, ouabain (10 µM, and in [glucose]o-free or [Na(+]o-free conditions. CONCLUSIONS: Simvastatin causes ryanodine-sensitive Ca(2+ release which is important for AMPKα-Thr(172 phosphorylation via Ca(2+/CaMK II. AMPKα-Thr(172 phosphorylation causes [glucose]o uptake (and an [ATP]i increase, closure of KATP channels, and phosphorylation of AMPKα-Thr(172 and PP2A-Tyr(307 resulted. Phosphorylation of PP2A-Tyr(307 occurs at a site downstream of AMPKα-Thr(172 phosphorylation.

  2. Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

    Science.gov (United States)

    Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

    2014-11-01

    Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Cl−secretion in ATP-treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase A

    Science.gov (United States)

    Akimova, A Olga; Bourcier, Nathalie; Taurin, Sebastien; Bundey, Richard A; Grygorczyk, Konrad; Gekle, Michael; Insel, Paul A; Dulin, Nickolai O; Orlov, Sergei N

    2005-01-01

    This study examines the mechanism of P2Y-induced Cl− secretion in monolayers of C7–Madin–Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (ISC). Both ATP-induced arachidonic acid (AA) synthesis and ISC in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased ISC and cAMP production in ATP-treated cells with an IC50 of ∼0.3 μm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and ISC evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and ISC triggered by ATP and AA, but did not affect PKA activation and ISC detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Cai2+ chelator BAPTA. Neither baseline nor maximal increment of ATP-induced ISC was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P2Y1 and P2Y2 than for other P2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP ≫ UTP) indicates that P2Y1 rather than P2Y2 receptors contribute to PKA and ISC activation. Viewed collectively, these data show that Cl− secretion in C7–MDCK monolayers treated with basolateral ATP is triggered by P2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA. PMID:16109726

  4. Cl- secretion in ATP-treated renal epithelial C7-MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A2 and protein kinase A.

    Science.gov (United States)

    Akimova, A Olga; Bourcier, Nathalie; Taurin, Sebastien; Bundey, Richard A; Grygorczyk, Konrad; Gekle, Michael; Insel, Paul A; Dulin, Nickolai O; Orlov, Sergei N

    2005-11-01

    This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP > UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

  5. H(2) mediates cardioprotection via involvements of K(ATP) channels and permeability transition pores of mitochondria in dogs.

    Science.gov (United States)

    Yoshida, Akemi; Asanuma, Hiroshi; Sasaki, Hideyuki; Sanada, Shoji; Yamazaki, Satoru; Asano, Yoshihiro; Shinozaki, Yoshiro; Mori, Hidezo; Shimouchi, Akito; Sano, Motoaki; Asakura, Masanori; Minamino, Tetsuo; Takashima, Seiji; Sugimachi, Masaru; Mochizuki, Naoki; Kitakaze, Masafumi

    2012-06-01

    Inhalation of hydrogen (H(2)) gas has been shown to limit infarct size following ischemia-reperfusion injury in rat hearts. However, H(2) gas-induced cardioprotection has not been tested in large animals and the precise cellular mechanism of protection has not been elucidated. We investigated whether opening of mitochondrial ATP-sensitive K+ channels (mK(ATP)) and subsequent inhibition of mitochondrial permeability transition pores (mPTP) mediates the infarct size-limiting effect of H(2) gas in canine hearts. The left anterior descending coronary artery of beagle dogs was occluded for 90 min followed by reperfusion for 6 h. Either 1.3% H(2) or control gas was inhaled from 10 min prior to start of reperfusion until 1 h of reperfusion, in the presence or absence of either 5-hydroxydecanoate (5-HD; a selective mK(ATP) blocker), or atractyloside (Atr; a mPTP opener). Systemic hemodynamic parameters did not differ among the groups. Nevertheless, H(2) gas inhalation reduced infarct size normalized by risk area (20.6±2.8% vs. control gas 44.0±2.0%; pchannels followed by inhibition of mPTP. H(2) gas may provide an effective adjunct strategy in patients with acute myocardial infarction receiving reperfusion therapy.

  6. Local release of ATP into the arterial inflow and venous drainage of human skeletal muscle: insight from ATP determination with the intravascular microdialysis technique

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Thaning, Pia; Nyberg, Michael Permin

    2011-01-01

    is released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous...

  7. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Science.gov (United States)

    Chan, Grace Ka Yan; Kleinheinz, Tracy L; Peterson, David; Moffat, John G

    2013-01-01

    In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  8. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

    Science.gov (United States)

    Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

    2017-05-26

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

    DEFF Research Database (Denmark)

    Beuschlein, Felix; Boulkroun, Sheerazed; Osswald, Andrea

    2013-01-01

    Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α...... subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced...... affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation...

  10. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...

  11. Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

    Science.gov (United States)

    Theler, J M; Mollard, P; Guérineau, N; Vacher, P; Pralong, W F; Schlegel, W; Wollheim, C B

    1992-09-05

    In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still

  12. Modeling the effects of hypoxia on ATP turnover in exercising muscle

    Science.gov (United States)

    Arthur, P. G.; Hogan, M. C.; Bebout, D. E.; Wagner, P. D.; Hochachka, P. W.

    1992-01-01

    Most models of metabolic control concentrate on the regulation of ATP production and largely ignore the regulation of ATP demand. We describe a model, based on the results of Hogan et al. (J. Appl. Physiol. 73: 728-736, 1992), that incorporates the effects of ATP demand. The model is developed from the premise that a unique set of intracellular conditions can be measured at each level of ATP turnover and that this relationship is best described by energetic state. Current concepts suggest that cells are capable of maintaining oxygen consumption in the face of declines in the concentration of oxygen through compensatory changes in cellular metabolites. We show that these compensatory changes can cause significant declines in ATP demand and result in a decline in oxygen consumption and ATP turnover. Furthermore we find that hypoxia does not directly affect the rate of anaerobic ATP synthesis and associated lactate production. Rather, lactate production appears to be related to energetic state, whatever the PO2. The model is used to describe the interaction between ATP demand and ATP supply in determining final ATP turnover.

  13. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes.

    Science.gov (United States)

    Burkeen, Jeff F; Womac, Alisa D; Earnest, David J; Zoran, Mark J

    2011-06-08

    The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca(2+) and ATP accumulation and have demonstrated that intracellular Ca(2+) levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca(2+) levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca(2+). In contrast, our finding that peak mitochondrial Ca(2+) occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca(2+) uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca(2+) mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

  14. ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology.

    Science.gov (United States)

    De Col, Valentina; Fuchs, Philippe; Nietzel, Thomas; Elsässer, Marlene; Voon, Chia Pao; Candeo, Alessia; Seeliger, Ingo; Fricker, Mark D; Grefen, Christopher; Møller, Ian Max; Bassi, Andrea; Lim, Boon Leong; Zancani, Marco; Meyer, Andreas J; Costa, Alex; Wagner, Stephan; Schwarzländer, Markus

    2017-07-18

    Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

  15. Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner

    Science.gov (United States)

    Hironaka, Ippei; Iwase, Tadayuki; Sugimoto, Shinya; Okuda, Ken-ichi; Tajima, Akiko; Yanaga, Katsuhiko

    2013-01-01

    ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 μM/108 cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function. PMID:23354720

  16. Silencing of atp6v1c1 prevents breast cancer growth and bone metastasis.

    Science.gov (United States)

    Feng, Shengmei; Zhu, Guochun; McConnell, Matthew; Deng, Lianfu; Zhao, Qiang; Wu, Mengrui; Zhou, Qi; Wang, Jinshen; Qi, Jin; Li, Yi-Ping; Chen, Wei

    2013-01-01

    Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.

  17. Activation of ATP/UTP-selective receptors increases blood flow and blunts sympathetic vasoconstriction in human skeletal muscle

    DEFF Research Database (Denmark)

    Yegutkin, G.G.; Gonzalez-Alonso, J.; Rosenmeier, Jaya Birgitte

    2008-01-01

    and sympatholytic effects of exogenous ATP in the skeletal muscle vasculature are largely mediated via ATP itself rather than its dephosphorylated metabolites, most likely via binding to endothelial ATP/UTP-selective P2Y(2) receptors. These data are consistent with a role of ATP in skeletal muscle hyperaemia...

  18. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    NARCIS (Netherlands)

    Tjaden, J.; Haferkamp, I.; Boxma, B.; Tielens, A.G.; Huynen, M.A.; Hackstein, J.H.P.

    2004-01-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and

  19. Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

    DEFF Research Database (Denmark)

    Johansen, Torben; Praetorius, Birger Hans

    1994-01-01

    The Na+/K(+)-pump activity and the utilization of adenosine triphosphate (ATP) were studied in rat peritoneal mast cells after histamine secretion induced by compound 48/80. We measured the ouabain-sensitive K(+)-uptake by a radioactive technique (86Rb+). The ATP content and the glycolytic ATP...

  20. Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.

    Directory of Open Access Journals (Sweden)

    Tina Rönn

    Full Text Available BACKGROUND: Impaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D. Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms (SNPs and risk of T2D. METHODOLOGY/PRINCIPAL FINDINGS: ATP5O mRNA expression was analyzed in skeletal muscle from young (n = 86 and elderly (n = 68 non-diabetic twins before and after a hyperinsulinemic euglycemic clamp. 11 SNPs from the ATP5O locus were genotyped in the twins and a T2D case-control cohort (n = 1466. DNA methylation of the ATP5O promoter was analyzed in twins (n = 22 using bisulfite sequencing. The mRNA level of ATP5O in skeletal muscle was reduced in elderly compared with young twins, both during basal and insulin-stimulated conditions (p<0.0005. The degree of DNA methylation around the transcription start of ATP5O was <1% in both young and elderly twins and not associated with mRNA expression (p = 0.32. The mRNA level of ATP5O in skeletal muscle was positively related to insulin-stimulated glucose uptake (regression coefficient = 6.6; p = 0.02. Furthermore, two SNPs were associated with both ATP5O mRNA expression (rs12482697: T/T versus T/G; p = 0.02 and rs11088262: A/A versus A/G; p = 0.004 and glucose uptake (rs11088262: A/A versus A/G; p = 0.002 and rs12482697: T/T versus T/G; p = 0.005 in the young twins. However, we could not detect any genetic association with T2D. CONCLUSIONS/SIGNIFICANCE: Genetic variation and age are associated with skeletal muscle ATP5O mRNA expression and glucose disposal rate, suggesting that combinations of genetic and non-genetic factors may cause the reduced expression of ATP5O in T2D muscle. These findings propose a role for ATP5O, in

  1. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    Science.gov (United States)

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  2. Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

    Science.gov (United States)

    Park, Yoojin; Nim-Anussornkul, Duangrat; Vilaivan, Tirayut; Morii, Takashi; Kim, Byeang Hyean

    2018-01-15

    We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

    LENUS (Irish Health Repository)

    Pitceathly, Robert D S

    2012-09-11

    Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

  4. Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

    DEFF Research Database (Denmark)

    Liu, Jianming; Kandasamy, Vijayalakshmi; Würtz, Anders

    2016-01-01

    Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase into a Lact......Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase...... mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream...

  5. The cotton ATP synthase δ1 subunit is required to maintain a higher ATP/ADP ratio that facilitates rapid fibre cell elongation.

    Science.gov (United States)

    Pang, Y; Wang, H; Song, W-Q; Zhu, Y-X

    2010-11-01

    The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthase activity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors of ATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation. © 2010 German Botanical Society and The Royal Botanical Society of The Netherlands.

  6. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants

    OpenAIRE

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, includ...

  7. Treatment with Oral ATP decreases alternating hemiplegia of childhood with de novo ATP1A3 Mutation.

    Science.gov (United States)

    Ju, Jun; Hirose, Shinichi; Shi, Xiu-Yu; Ishii, Atsushi; Hu, Lin-Yan; Zou, Li-Ping

    2016-05-04

    Alternating hemiplegia of childhood is an intractable neurological disorder characterized by recurrent episodes of alternating hemiplegia accompanied by other paroxysmal symptoms. Recent research has identified mutations in the ATP1A3 gene as the underlying cause. Adenosine-5'-triphosphate has a vasodilatory effect, can enhance muscle strength and physical performance, and was hypothesized to improve the symptoms of paroxysmal hemiplegia. A 7-year-old boy with alternating hemiplegia of childhood who was positive for a de novo ATP1A3 mutation was treated with adenosine- 5'- triphosphate supplementation orally as an innovative therapy for 2 years. Outcome was evaluated through the follow-up of improvement of hemiplegic episodes and psychomotor development. Side effects and safety were monitored in regularity. With the dosage of adenosine-5'-triphosphate administration increased, the patient showed significantly less frequency and shorter duration of hemiplegic episodes. Treatment with adenosine-5'-triphosphate was correlated with a marked amelioration of alternating hemiplegia of childhood episodes, and psychomotor development has improved. The maximum dose of oral administration of adenosine-5'-triphosphate reached 25 mg/kg per day. Adenosine-5'-triphosphate therapy was well tolerated without complaint of discomfort and side effects. The 2-year follow-up outcome of adenosine-5'-triphosphate therapy for alternating hemiplegia of childhood was successful.

  8. The modulation of vascular ATP-sensitive K+ channel function via the phosphatidylinositol 3-kinase-Akt pathway activated by phenylephrine.

    Science.gov (United States)

    Haba, Masanori; Hatakeyama, Noboru; Kinoshita, Hiroyuki; Teramae, Hiroki; Azma, Toshiharu; Hatano, Yoshio; Matsuda, Naoyuki

    2010-08-01

    The present study examined the modulator role of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway activated by the alpha-1 adrenoceptor agonist phenylephrine in ATP-sensitive K(+) channel function in intact vascular smooth muscle. We evaluated the ATP-sensitive K(+) channel function and the activity of the PI3K-Akt pathway in the rat thoracic aorta without endothelium. The PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (10(-5) M) augmented relaxation in response to the ATP-sensitive K(+) channel opener levcromakalim (10(-8) to 3 x 10(-6) M) in aortic rings contracted with phenylephrine (3 x 10(-7) M) but not with 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619; 3 x 10(-8) M), although those agents induced similar contraction. ATP-sensitive K(+) channel currents induced by levcromakalim (10(-6) M) in the presence of phenylephrine (3 x 10(-7) M) were enhanced by the nonselective alpha-adrenoceptor antagonist phentolamine (10(-7) M) and LY294002 (10(-5) M). Levels of the regulatory subunits of PI3K p85-alpha and p55-gamma increased in the membrane fraction from aortas without endothelium treated with phenylephrine (3 x 10(-7) M) but not with U46619 (3 x 10(-8) M). Phenylephrine simultaneously augmented Akt phosphorylation at Ser473 and Thr308. Therefore, activation of the PI3K-Akt pathway seems to play a role in the impairment of ATP-sensitive K(+) channel function in vascular smooth muscle exposed to alpha-1 adrenergic stimuli.

  9. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  10. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou (NCSU)

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  11. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa.

    Science.gov (United States)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

    2016-10-01

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space group C222 or C2221, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  12. Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens.

    Science.gov (United States)

    Haddix, Pryce L; Jones, Sarah; Patel, Pratik; Burnham, Sarah; Knights, Kaori; Powell, Joan N; LaForm, Amber

    2008-11-01

    Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.

  13. Docosahexaenoic acid and L-Carnitine prevent ATP loss in SH-SY5Y neuroblastoma cells after exposure to silver nanoparticles.

    Science.gov (United States)

    Tan, Joey Wee-Shan; Ho, Christabel Fung-Yih; Ng, Yee-Kong; Ong, Wei-Yi

    2016-02-01

    Silver nanoparticles (AgNPs) are among the most commonly used nanomaterials, but thus far, little is known about ways to mitigate against potential toxic effects of exposure. In this study, we examined the potential effects of AgNPs on mitochondrial function and cellular ATP levels, and whether these could be prevented by treatment with docosahexaenoic acid (DHA) and L-carnitine (LC). Acute exposure of AgNPs for 1 h to SH-SY5Y cells resulted in decreased mitochondrial membrane potential, and decreased ATP and ADP levels, indicating mitochondrial damage and reduced production of ATP. Incubation of cells with DHA partially reduced, while treatment with LC and DHA completely abolished the AgNP induced decreases in ATP and ADP levels. This could be due to a LC-facilitated entry of DHA to mitochondria, for repair of damaged phospholipids. It is postulated that DHA and LC may be useful for treatment of accidental environmental exposure to AgNPs. © 2014 Wiley Periodicals, Inc.

  14. NASA ATP Force Measurement Technology Capability Strategic Plan

    Science.gov (United States)

    Rhew, Ray D.

    2008-01-01

    The Aeronautics Test Program (ATP) within the National Aeronautics and Space Administration (NASA) Aeronautics Research Mission Directorate (ARMD) initiated a strategic planning effort to re-vitalize the force measurement capability within NASA. The team responsible for developing the plan included members from three NASA Centers (Langley, Ames and Glenn) as well as members from the Air Force s Arnold Engineering and Development Center (AEDC). After visiting and discussing force measurement needs and current capabilities at each participating facility as well as selected force measurement companies, a strategic plan was developed to guide future NASA investments. This paper will provide the details of the strategic plan and include asset management, organization and technology research and development investment priorities as well as efforts to date.

  15. Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes

    Science.gov (United States)

    Hong, Miyoun; Bao, Li; Kefaloyianni, Eirini; Agullo-Pascual, Esperanza; Chkourko, Halina; Foster, Monique; Taskin, Eylem; Zhandre, Marine; Reid, Dylan A.; Rothenberg, Eli; Delmar, Mario; Coetzee, William A.

    2012-01-01

    Ventricular ATP-sensitive potassium (KATP) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative KATP channel-associated proteins. We investigated whether the association of KATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between KATP channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that KATP channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that KATP channels are clustered within nanometer distances from junctional proteins. The local KATP channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The KATP channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell KATP channel current density (activated by metabolic inhibition) was ∼40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of KATP channels was absent in the PKP2 deficient mice, but the KATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of KATP channel distribution within a cardiac myocyte. The higher KATP channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia. PMID:23066018

  16. Cervical anterior transpedicular screw fixation (ATPS)—Part II. Accuracy of manual insertion and pull-out strength of ATPS

    National Research Council Canada - National Science Library

    Koller, Heiko; Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H; Resch, Herbert; Hitzl, Wolfgang

    2008-01-01

    .... To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation...

  17. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential......The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle...... and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content...

  18. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  19. Antispasmodic activity of Symplocos paniculata is mediated through opening of ATP-dependent K+ channel

    Directory of Open Access Journals (Sweden)

    Khalid Hussain Janbaz

    2016-06-01

    Full Text Available Symplocos paniculata is a medicinal plant used by native healers to manage gastrointestinal ailments. The crude methanolic extract of S. paniculata was screened pharmacologically both in vitro and in vivo for the validation of its therapeutic potential. It suppressed the spontaneous activity of isolated rabbit jejunum preparations and also caused inhibition of the low K+ (20 mM- induced spastic contractions in isolated rabbit jejunum preparations in a manner comparable to cromakalim. The relaxant effect was found to be blocked following glibenclamide exposure of the isolated tissue preparations similar to cromakalim, suggesting that observed response was likely to be mediated through opening of ATP dependent K+ channels. Following oral administration to mice provided protection against castor oil-induced diarrhea in a manner similar to loperamide. The plant material was found safe in toxicity study up to oral dose of 8 g/kg in mice. Hence, present study provides a scientific basis for the vernacular use of S. paniculata in gastro-intestinal system.

  20. Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

    2012-01-01

    Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

  1. Global gene profiling of aging lungs in Atp8b1 mutant mice.

    Science.gov (United States)

    Soundararajan, Ramani; Stearns, Timothy M; Czachor, Alexander; Fukumoto, Jutaro; Turn, Christina; Westermann-Clark, Emma; Breitzig, Mason; Tan, Lee; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

    2016-09-29

    Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes. We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR). Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs. Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases.

  2. ATP Binding and Hydrolysis Properties of ABCB10 and Their Regulation by Glutathione

    Science.gov (United States)

    Qiu, Wei; Liesa, Marc; Carpenter, Elizabeth P.; Shirihai, Orian S.

    2015-01-01

    ABCB10 (ATP binding cassette sub-family B10) is a mitochondrial inner-membrane ABC transporter. ABCB10 has been shown to protect the heart from the impact of ROS during ischemia-reperfusion and to allow for proper hemoglobin synthesis during erythroid development. ABC transporters are proteins that increase ATP binding and hydrolysis activity in the presence of the transported substrate. However, molecular entities transported by ABCB10 and its regulatory mechanisms are currently unknown. Here we characterized ATP binding and hydrolysis properties of ABCB10 by using the 8-azido-ATP photolabeling technique. This technique can identify potential ABCB10 regulators, transported substrates and amino-acidic residues required for ATP binding and hydrolysis. We confirmed that Gly497 and Lys498 in the Walker A motif, Glu624 in the Walker B motif and Gly602 in the C-Loop motif of ABCB10 are required for proper ATP binding and hydrolysis activity, as their mutation changed ABCB10 8-Azido-ATP photo-labeling. In addition, we show that the potential ABCB10 transported entity and heme precursor delta-aminolevulinic acid (dALA) does not alter 8-azido-ATP photo-labeling. In contrast, oxidized glutathione (GSSG) stimulates ATP hydrolysis without affecting ATP binding, whereas reduced glutathione (GSH) inhibits ATP binding and hydrolysis. Indeed, we detectABCB10 glutathionylation in Cys547 and show that it is one of the exposed cysteine residues within ABCB10 structure. In all, we characterize essential residues for ABCB10 ATPase activity and we provide evidence that supports the exclusion of dALA as a potential substrate directly transported by ABCB10. Last, we show the first molecular mechanism by which mitochondrial oxidative status, through GSH/GSSG, can regulate ABCB10. PMID:26053025

  3. Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue.

    Science.gov (United States)

    Balaz, Martina; Sundberg, Mark; Persson, Malin; Kvassman, Jan; Månsson, Alf

    2007-06-19

    Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.

  4. Glycolysis-related gene induction and ATP reduction during fractionated irradiation. Markers for radiation responsiveness of human tumor xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Goetze, K.; Meyer, S.S.; Mueller-Klieser, W. [University Medical Center Mainz Univ. (Germany). Inst. of Physiology and Pathophysiology; Yaromina, A. [Technical Univ. Dresden (Germany). OncoRay - National Center for Radiation Research in Oncology; Zips, D. [University Hospital Tuebingen (Germany). Dept. of Radiation Oncology; Baumann, M. [Technical Univ. Dresden (Germany). OncoRay - National Center for Radiation Research in Oncology; University Hospital Dresden Technical Univ. Dresden (Germany). Dept. of Radiation Oncology

    2013-09-15

    Background and purpose: Lactate was previously shown to be a prognostic but not a predictive pre-therapeutic marker for radiation response of tumor xenografts. We hypothesize that metabolic changes during fractionated irradiation may restrict the predictiveness of lactate regarding tumor radiosensitivity. Materials and methods: Tumor xenografts were generated in nude mice by implanting 4 head and neck squamous cell carcinoma lines with different sensitivities to fractionated irradiation. Tumors were irradiated with up to 15 fractions of 2 Gy over a period of 3 weeks, and ATP and lactate levels were measured in vital tumor areas with induced metabolic bioluminescence imaging. Corresponding changes in mRNA expression of glycolysis-related genes were determined by quantitative RT-PCR. Results: Lactate content decreased significantly in 3 out of 4 cell lines in the course of irradiation showing no correlation with cell line-specific radiosensitivity. Radiation-induced changes in ATP levels and glycolysis-related mRNA expression, however, only occurred in radiosensitive or intermediately radioresistant xenografts, whereas these parameters remained unchanged in radioresistant tumors. Conclusion: Sensitivity-related differences in the transcriptional response of tumors to radiotherapy may be exploited in the clinic for better individualization of tumor treatment. (orig.)

  5. RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner.

    Science.gov (United States)

    Peisley, Alys; Wu, Bin; Yao, Hui; Walz, Thomas; Hur, Sun

    2013-09-12

    Retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are paralogous receptors for viral double-stranded RNA (dsRNA) with divergent specificity. We have previously shown that MDA5 forms filaments upon viral dsRNA recognition and that this filament formation is essential for interferon signal activation. Here, we show that while RIG-I binds to a dsRNA end as a monomer in the absence of ATP, it assembles in the presence of ATP into a filament that propagates from the dsRNA end to the interior. Furthermore, RIG-I filaments directly stimulate mitochondrial antiviral signaling (MAVS) filament formation without any cofactor, such as polyubiquitin chains, and forced juxtaposition of the isolated signaling domain of RIG-I, as it would be in the filament, is sufficient to activate interferon signaling. Our findings thus define filamentous architecture as a common yet versatile molecular platform for divergent viral RNA detection and proximity-induced signal activation by RIG-I and MDA5. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Development of an ATP measurement method suitable for xenobiotic treatment activated sludge biomass.

    Science.gov (United States)

    Nguyen, Lan Huong; Chong, Nyuk-Min

    2015-09-01

    Activated sludge consumes a large amount of energy to degrade a xenobiotic organic compound. By tracking the energy inventory of activated sludge biomass during the sludge's degradation of a xenobiotic, any disadvantageous effect on the sludge's performance caused by energy deficiency can be observed. The purpose of this study was to develop a reliable and accurate method for measuring the ATP contents of activated sludge cells that were to degrade a xenobiotic organic. Cell disruption and cellular ATP extraction were performed by a protocol with which xenobiotic degrading activated sludge biomass was washed with SDS, treated by Tris and TCA, and followed by bead blasting. The suspension of disrupted cells was filtered before the filtrate was injected into HPLC that was set at optimal conditions to measure the ATP concentration therein. This extraction protocol and HPLC measurement of ATP was evaluated for its linearity, limits of detection, and reproducibility. Evaluation test results reported a R(2) of 0.999 of linear fit of ATP concentration versus activated sludge concentration, a LOD=0.00045mg/L, a LOQ=0.0015mg/L for HPLC measurement of ATP, a MDL=0.46mg/g SS for ATP extraction protocol, and a recovery efficiency of 96.4±2%. This method of ATP measurement was simple, rapid, reliable, and was unburdened of some limitations other methods may have. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels.

    NARCIS (Netherlands)

    Carrasco, A.J.; Dzeja, P.P.; Alekseev, A.E.; Pucar, D.; Zingman, L.V.; Abraham, M.R.; Hodgson, D.M.; Bienengraeber, M.; Puceat, M.; Janssen, E.E.W.; Wieringa, B.; Terzic, A.

    2001-01-01

    Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP))

  8. Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

    NARCIS (Netherlands)

    Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

    2016-01-01

    Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in

  9. A futile cycle, formed between two ATP-dependant γ-glutamyl cycle ...

    Indian Academy of Sciences (India)

    Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the ...

  10. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

  11. Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A.; Xing, Yongna

    2013-10-08

    Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.

  12. An Aptamer-Based Nanobiosensor for Real-Time Measurements of ATP Dynamics

    DEFF Research Database (Denmark)

    Özalp, Cengiz; Nielsen, Lise Junker; Olsen, Lars Folke

    2010-01-01

    A nanosensor based on a new DNA aptamer can show changes in intracellular concentration of ATP of 0.5 to 8 mM and thereby the kinetics of ATP-consuming reactions in real-time. The biosensor was protected against nuclease attack by being buried in a polyacrylamide nanoparticle. This strategy can...

  13. Small amounts of functional ATP7A protein permit mild phenotype

    DEFF Research Database (Denmark)

    Møller, Lisbeth Birk

    2015-01-01

    in the ATP7A gene. We have conducted several studies in the hope of uncovering the relationship between genotype and phenotype. We have examined the X-inactivation pattern in affected females, the effect of exon-deletions and-duplications, and splice-site mutations on the composition and amount of ATP7A...... individuals exhibited preferential inactivation of the X-chromosome with the mutated ATP7A gene. The observed mild phenotype in some patients with mutations that effect the composition of the ATP7A transcript, seems to be explained by the presence of a small amount of normal ATP7A transcript. The location...... of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with copper, whereas at higher...

  14. Energetic and metabolic consequences of aerobic and an-aerobic ATP-production.

    NARCIS (Netherlands)

    Schreurs, V.V.A.M.; Aarts, M.J.; IJssennagger, N.; Hermans, J.; Hendriks, W.H.

    2007-01-01

    ATP, the currency of cellular energy metabolism, can be produced during aerobic and an-aerobic oxidation of metabolic substrates. The aerobic oxidation yields CO2 + H2O as metabolic end products while ATP is produced by oxidative phosphorylation in the mitochondria. Carbohydrate, protein and fat

  15. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  16. Effect of visible laser light on ATP level of anaemic red blood cell.

    Science.gov (United States)

    Suardi, Nursakinah; Sodipo, Bashiru Kayode; Mustafa, Mohd Zulkifli; Ali, Zalila

    2016-09-01

    In this work we present influence of visible laser light on ATP level and viability of anaemic red blood cell (RBC). The visible laser lights used in this work are 460nm and 532nm. The responses of ATP level in anaemic and normal RBC before and after laser irradiation at different exposure time (30, 40, 50 and 60s) were observed. Three aliquots were prepared from the ethylenediaminetetraacetic acid (EDTA) blood sample. One served as a control (untreated) and another two were irradiated with 460nm and 560nm lasers. Packed RBC was prepared to study ATP level in the RBC using CellTiter-GloLuminescent cell Viability Assay kit. The assay generates a glow type signal produced by luciferase reaction, which is proportional to the amount of ATP present in RBCs. Paired t-test were done to analyse ATP level before and after laser irradiation. The results revealed laser irradiation improve level of ATP in anaemic RBC. Effect of laser light on anaemic RBCs were significant over different exposure time for both 460nm (p=0.000) and 532nm (p=0.003). The result of ATP level is further used as marker for RBC viability. The influence of ATP level and viability were studied. Optical densities obtained from the data were used to determine cell viability of the samples. Results showed that laser irradiation increased viability of anaemic RBC compared to normal RBC. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  18. Protein Phosphorylation and Prevention of Cytochrome Oxidase Inhibition by ATP: Coupled Mechanisms of Energy Metabolism Regulation

    National Research Council Canada - National Science Library

    Acin-Perez, Rebeca; Gatti, Domenico L; Bai, Yidong; Manfredi, Giovanni

    2011-01-01

    ... cytochrome oxidase (COX), the terminal enzyme of the electron transfer chain (ETC), as one of its targets ( Acin-Perez et al., 2009a, 2009b ). COX translocates protons across the inner mitochondrial membrane, contributing to the proton electrochemical gradient used by the ATP synthase to generate ATP. In living cells, COX is a pacemaker ...

  19. The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

    Science.gov (United States)

    Forsyth, Alison; Abkarian, Manouk; Wan, Jiandi; Stone, Howard

    2010-11-01

    In shear flow, red blood cells (RBCs) exhibit a variety of behaviors such as rouleaux formation, tumbling, swinging, and tank-treading. The physiological consequences of these dynamic behaviors are not understood. In vivo, ATP is known to signal vasodilation; however, to our knowledge, no one has deciphered the relevance of RBC microrheology to the functional release of ATP. Previously, we correlated RBC deformation and ATP release in microfluidic constrictions (Wan et al., 2008). In this work, a cone-plate rheometer is used to shear a low hematocrit solution of RBCs at varying viscosity ratios (λ) between the inner cytoplasmic hemoglobin and the outer medium, to determine the intrinsic viscosity of the suspension. Further, using a luciferin-luciferase enzymatic reaction, we report the relative ATP release at varying shear rates. Results indicate that for λ = 1.6, 3.8 and 11.1, ATP release is constant up to 500 s-1, which suggests that the tumbling-tanktreading transition does not alter ATP release in pure shear. For lower viscosity ratios, λ = 1.6 and 3.8, at 500 s-1 a change in slope occurs in the intrinsic viscosity data and is marked by an increase in ATP release. Based on microfluidic observations, this simultaneous change in viscosity and ATP release occurs within the tank-treading regime.

  20. Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

    Directory of Open Access Journals (Sweden)

    Askew Christopher D

    2009-12-01

    Full Text Available Abstract Background It has been proposed that adenosine triphosphate (ATP released from red blood cells (RBCs may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC. Results Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P Conclusions Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

  1. Towards the development of an automated ATP measuring platform to monitor microbial quality of drinking water

    DEFF Research Database (Denmark)

    Tatari, Karolina; Hansen, C. B.; Rasmussen, A.

    This work aimed to develop an automated and nearly on-line method to monitor ATP levels in drinking water as an indicator of microbial contamination. The system consists of a microfluidic cartridge installed in a light tight box, where the sample is mixed with the reagents and the emitted light...... is detected by a photomultiplier. Temperature in the assay box is controlled and set to 25°C. Calibration of the system using ATP standard solutions was successful, both for free and for total ATP. Chemical release of ATP by reagent addition however resulted in the formation of particles that ultimately...... clogged the microfluidic channels. An alternative thermal lysis step was implemented, by adding a flow-though heating/cooling step to the system. Thermal lysis showed efficient release of ATP from an E. coli dilution, but the releasing efficiency varied according to the type of water. Overall...

  2. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    Science.gov (United States)

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-01-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

  3. Structural and metabolic changes in Atp7b-/- mouse liver and potential for new interventions in Wilson's disease.

    Science.gov (United States)

    Huster, Dominik

    2014-05-01

    Wilson's disease (WD) is caused by ATP7B mutations and results in copper accumulation and toxicity in liver and brain tissues. The specific mechanisms underlying copper toxicity are still poorly understood. Mouse models have revealed new insights into pathomechanisms of hepatic WD. Mitochondrial damage is observed in livers of WD patients and in mouse models; copper induces fragmentation of mitochondrial membrane lipids, particularly cardiolipin, with deleterious effects on both mitochondrial integrity and function. Copper accumulation also induces chronic inflammation in WD livers, which is followed by regeneration in parts of the liver and occasionally neoplastic proliferation. Gene expression studies using microarrays have aided our understanding of the molecular basis of these changes. Copper overload alters cholesterol biosynthesis in hepatocytes resulting in reduced liver and serum cholesterol. Experiments are currently underway to elucidate the link between copper and cholesterol metabolism. These findings may facilitate the development of specific therapies to ameliorate WD progression. © 2014 New York Academy of Sciences.

  4. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    Directory of Open Access Journals (Sweden)

    Reedijk Jan

    2008-06-01

    Full Text Available Abstract Background Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Methods Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of Results In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Conclusion Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours

  5. Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2.

    Science.gov (United States)

    Hosokawa, Yuki; Takahashi, Hisaaki; Inoue, Akihiro; Kawabe, Yuya; Funahashi, Yu; Kameda, Kenji; Sugimoto, Kana; Yano, Hajime; Harada, Hironobu; Kohno, Shohei; Ohue, Shiro; Ohnishi, Takanori; Tanaka, Junya

    2015-06-01

    Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Ca++-sensitizing mutations in troponin, Pi, and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

    Science.gov (United States)

    Longyear, Thomas J.; Turner, Matthew A.; Davis, Jonathan P.; Lopez, Joseph; Biesiadecki, Brandon

    2014-01-01

    Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca++-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity (VRTF) at both maximal and submaximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn (TnC) increased VRTF at submaximal Ca++ under acidic conditions but had no effect on VRTF at maximal Ca++ levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate. PMID:24651988

  7. Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

    Science.gov (United States)

    Shibukawa, Yoshiyuki; Sato, Masaki; Kimura, Maki; Sobhan, Ubaidus; Shimada, Miyuki; Nishiyama, Akihiro; Kawaguchi, Aya; Soya, Manabu; Kuroda, Hidetaka; Katakura, Akira; Ichinohe, Tatsuya; Tazaki, Masakazu

    2015-04-01

    Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

  8. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Identification of a site of ATP requirement for signal processing in bacterial chemotaxis.

    Science.gov (United States)

    Smith, J M; Rowsell, E H; Shioi, J; Taylor, B L

    1988-06-01

    In Escherichia coli and Salmonella typhimurium, ATP is required for chemotaxis and for a normal probability of clockwise rotation of the flagellar motors, in addition to the requirement for S-adenosylmethionine (J. Shioi, R. J. Galloway, M. Niwano, R. E. Chinnock, and B. L. Taylor, J. Biol. Chem. 257:7969-7975, 1982). The site of the ATP requirement was investigated. The times required for S. typhimurium ST23 (hisF) to adapt to a step increase in serine, phenol, or benzoate were similar in cells depleted of ATP and in cells with normal levels of ATP. This established that ATP was not required for the chemotactic signal to cross the inner membrane or for adaptation to the transmembrane signal to occur. Depletion of ATP did not affect the probability of clockwise rotation in E. coli cheYZ scy strains that were defective in the cheY and cheZ genes and had a partially compensating mutation in the motor switch. Strain HCB326 (cheAWRBYZ tar tap tsr trg::Tn10), which was deficient in all chemotaxis components except the switch and motor, was transformed with the pCK63 plasmid (ptac-cheY+). Induction of cheY in the transformant increased the frequency of clockwise rotation, but except at the highest levels of CheY overproduction, clockwise rotation was abolished by depleting ATP. It is proposed that the CheY protein is normally in an inactive form and that ATP is required for formation of an active CheY* protein that binds to the switch on the flagellar motors and initiates clockwise rotation. Depletion of ATP partially inhibits feedback regulation of the cheB product, protein methylesterase, but this may reflect a second site of ATP action in chemotaxis.

  10. Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eBohrer

    2015-01-01

    Full Text Available Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS is the enzyme catalyzing the key entr