Sample records for ca2 mg2 atpase

  1. Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase.

    Cavieres, J D


    Calmodulin-depleted red cell membranes catalyse a Ca2+, Mg2+-dependent ATP-[3H]ADP exchange at 37 degrees C. The Ca2+, Mg2+-dependent exchange, measured at 20 microM CaCl2, 1.5 mM MgCl2, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca2+ + Mg2+)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. EDTA-washed membranes present a Ca2+-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg2+-containing medium, while their Ca2+-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl2 to the medium restores both activities to the levels found with membranes not treated with EDTA. Calmodulin (16 micrograms/ml) produces an eight-fold stimulation of the Ca2+-dependent ATP-ADP exchange, slightly less than it stimulates the Ca2+-dependent ATP hydrolysis. The effect of 1.5 mM MgCl2 on the exchange is greater in the presence than in the absence of calmodulin. It is proposed that the reversal of the initial phosphorylation of the Ca2+ pump, occurring at a fast rate at 37 degrees C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37 degrees C. The great difficulty in producing an overall reversal of the Ca2+ pump should then be due to one or more reaction steps later than and including Ca2+ release and dephosphorylation. PMID:2952171

  2. The influence of repeated irradiation of rats on activity of Ca2+ - ATPase and Mg2+ -ATPase in plasma membrane of thymocytes

    Rats were daily irradiated at doses 0.5 Gy in period of two weeks. The activity of Ca2+-ATPase Mg2+-ATPase and the extent of lipid peroxidation in thymus were determined. Thee peculiarity of changing of enzymes activity demonstrates the dependence of Mg2+-ATPase on lipid peroxidation

  3. Influence of phosphorylation of lymphocyte's plasma-membrane proteins and calmodulin on Ca2+, Mg2+ -ATPase activity under irradiation

    We establish that the regulation of Ca2+, Mg2+ - ATPase from plasma membranes of rat spleen lymphocytes is controlled by calmodulin and the Ca2+, calmodulin-dependent phosphorylation system. The mechanisms of regulation of this process are sensitive to the total X-ray irradiation in doses of 0.5 and 1 Gy

  4. Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts.

    Cavieres, J D


    An average target size of 251 kDa has been obtained for the (Ca2+ + Mg2+)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer. PMID:6142728

  5. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    Plasma membranes of human oat cell carcinoma possess Mg2+- and Ca2+-dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca2+-ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca2+-ATPase activity is more easily inactivated by Triton X-100, while the Mg2+-ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca2+-ATPase and Mg2+-ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [3H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca2+-ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg2+-ATPase has not been demonstrated, but is presently under investigation

  6. A phosphorylated conformational state of the (Ca2+-Mg2+)-ATPase of fast skeletal muscle sarcoplasmic reticulum can mediate rapid Ca2+ release.

    Chiesi, M; Wen, Y S


    A rapid Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles from fast skeletal muscle can be induced under conditions which permit the formation of a stable phosphorylated intermediate of the (Ca2+-Mg2+)-ATPase. Such a state can be achieved experimentally by phosphorylating the ATPase in the absence of Mg2+ ions, which otherwise would stimulate the dephosphorylation step(s). Also, quercetine stimulates the rapid release of Ca2+ if used in the concentration range which does not produce inhibition of phosphoenzyme formation, but which inhibits phosphoenzyme dephosphorylation. The rapid efflux of Ca2+ ions proceeds as long as the low affinity Ca2+-binding sites facing the lumen of the vesicles are saturated and as long as Ca2+ is removed from the catalytic sites facing the cytosol. A molecular mechanism of the phosphoenzyme-mediated Ca2+ release is proposed. This mechanism is based on a rapid shuttling of the ATPase molecules between an ADP-sensitive and an ADP-insensitive phosphorylated state. PMID:6133856

  7. Ca2+- and Mg2+-ATPase activities in winter wheat root plasma membranes as affected by NaCl stress during growth

    Mansour, MMF; van Hasselt, PR; Kuiper, PJC


    Winter wheat seedlings were grown in Hoagland nutrient solution with or without 100 mmol/L NaCl added. Plasma membranes from root cells were prepared by aqueous polymer two phase partitioning and the stimulation of plasma membrane ATPase activity by Mg2+ and Ca2+ was investigated. The enzyme was act


    Gunduluru Swathi


    Full Text Available Bacopa monnieri(BM; Family: Scrophulariaceae, also referred as Brahmi or Jalbrahmi has been used for centuries in Ayurvedic system of medicine as a brain tonic, memory enhancer, revitaliser of sensory organs, anti-anxiety, cardio-tonic, diuretic, antidepressant and anticonvulsant agent, and the pharmacological actions are mainly attributed to the saponin compounds present in the alcoholic extract of the plant. The present study was carried out with a specific aim to examine the neuroprotective effect of Bacopa monnieriduring Rotenone (RT induced Parkinson’s disease (PD with particular reference to Na+/K+, Mg2+and Ca2+-ATPase activities in different regions of rat brain. In the experiment conducted rats were divided into four groups of six in each group, group 1 received Salinewater (1 ml/kg, group 2 received RT (2.5 mg/kg through i.p. route administration for 60 days to induce PD. The third group received BM extract (180 mg/kg/day for 20 days orally before induction of PD and group 4 received Levodopa (LD (10 mg/kg/day orally which is referred as drug control. The levels of Na+/K+, Mg2+and Ca2+-ATPase activities were measured. Na+/K+, Mg2+and Ca2+-ATPase activities were significantly depleted in different brain regions of rat during RT induced PD when compared to control rats. Treatment with BM and LD caused significant elevation in the activity levels of Na+/K+, Mg2+and Ca2+-ATPase in different brain regions of rats when compared to induced PD rats. Our results suggest the ability of BM extract to modulate Na+/K+, Mg2+and Ca2+- ATPase activities in different brain regions of RT induced rodent model of PD and thus offers effective management in the treatment of PD.

  9. Effect of organic solvents on nervous cell membrane as measured by changes in the (Ca2+/Mg2+) ATPase activity and fluidity of synaptosomal membrane.

    Edelfors, S; Ravn-Jonsen, A


    The effect of various solvents on the central nervous system was studied by using rat brain synaptosomal membranes as an in vitro model. The activity of (Ca2+/Mg2+) ATPase and the membrane fluidity was determined. The alteration of the ATPase activity depended on the physio-chemical characteristics of the solvent in question. Incubation with aliphatic alkanes caused a stimulation of the ATPase activity whereas mixed hydrocarbons as kerosene, white spirit and gasoline inhibited the enzyme. Incubation with chlorinated hydrocarbons caused a biphasic response dependent on the concentration. Oxygen-containing hydrocarbons exhibited various effects as found after incubation with hydrocarbons. The different effects of the solvents on the ATPase activity suggest that the lipophilicity of the solvents is one of more parameters affecting the membrane. Furthermore, the biphasic response following the incubation with chlorinated hydrocarbons indicates that more mechanisms are involved in the enzyme effect. The membrane fluidity is increased with higher concentrations of the solvents. From the results it is concluded that the ATPase activity depends not only on the membrane fluidity and volume, but also on the hydrophilic vicinity of the enzyme molecule. PMID:1533717

  10. Effects of aqueous extract of Hibiscus sabdariffa on renal Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in Wistar rats%玫瑰茄提取物对Wistar大鼠肾Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性的影响

    Lawrence A.Olatunji; Taofeek O.Usman; Joseph O.Adebayo; Victoria A.Olatunji


    OBJECTIVE:To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in rats.METHODS:The 25 and 50 mg/(kg · d) of aqueous extracts of H.sabdariffa were respectively given to rats in the experimental groups for 28 d,and rats in the control group received an appropriate volume of distilled water as vehicle.Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in the kidney were assayed by spectrophotometric method. RESULTS:Administrations of 25 and 50 mg/(kg · d) of aqueous extract of H.sabdariffa significantly decreased the Ca2+-Mg2+-ATPase activity in the kidney of rats (P <0.05).However,the renal Na+-K+-ATpase activity of the experimental rats was not affected by either dose of the extract.And the plasma Na+,K+ and Ca2+ levels of the experimental rats had no significant changes.Administration of either dose of the extract did not result in any significant changes in body and kidney weights,the concentrations of plasma albumin and total protein,and alkaline phosphatase,aspartate aminotransferase and alanine aminotransferase activities.However,concentrations of creatinine and urea were significantly reduced by 50 mg/kg of the extract (P<0.05). CONCLUSION:The present study indicates that oral administration of aqueous extract of H.sabdariffa may preserve the renal function despite a decreased renal Ca2+-Mg2+-ATPase activity.%目的:研究口服玫瑰茄水提取物对大鼠肾Na+-K+-ATP酶和Ca2+- Mg2+-ATP酶活性的影响.方法:连续28 d分别给予实验大鼠口服25和50 mg/kg的玫瑰茄水提物,同时给予对照组大鼠灌胃适当剂量的蒸馏水.用光谱测定法分析大鼠肾脏中Na+-K+-ATP酶和Ca2+ -Mg2+-ATP酶的活性.结果:口服25和50 mg/kg的玫瑰茄提取物后,实验组大鼠肾Ca2+-Mg2+-ATP酶活性显著降低(P<0.05),然而肾Na+ -K+ -ATP酶的活性却未受到任何影响.实验组大鼠体质量、肾脏质量,血浆白蛋白和总蛋白浓

  11. Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats

    Yi-zong ZHAI; Chang-lin HUANG; Chang, Qi; Wang, Jiu-Qing; Zhang, Jia; Guo, Yan-Ling


    Objective To explore the effects of percutaneous impulsive current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats, in order to investigate the effect of exercise-induced fatigue. Methods Seventy-two 8-week old male Wistar rats were randomly divided into 4 groups (18 each): control group (group A), fatigue group (group B), stimulation before fatigue group (group C) and stimulation after fatigue group (...

  12. Effects of anthropogenic sound on digging behavior, metabolism, Ca2+/Mg2+ ATPase activity, and metabolism-related gene expression of the bivalve Sinonovacula constricta

    Peng, Chao; Zhao, Xinguo; Liu, Saixi; Shi, Wei; Han, Yu; Guo, Cheng; Jiang, Jingang; Wan, Haibo; Shen, Tiedong; Liu, Guangxu


    Anthropogenic sound has increased significantly in the past decade. However, only a few studies to date have investigated its effects on marine bivalves, with little known about the underlying physiological and molecular mechanisms. In the present study, the effects of different types, frequencies, and intensities of anthropogenic sounds on the digging behavior of razor clams (Sinonovacula constricta) were investigated. The results showed that variations in sound intensity induced deeper digging. Furthermore, anthropogenic sound exposure led to an alteration in the O:N ratios and the expression of ten metabolism-related genes from the glycolysis, fatty acid biosynthesis, tryptophan metabolism, and Tricarboxylic Acid Cycle (TCA cycle) pathways. Expression of all genes under investigation was induced upon exposure to anthropogenic sound at ~80 dB re 1 μPa and repressed at ~100 dB re 1 μPa sound. In addition, the activity of Ca2+/Mg2+-ATPase in the feet tissues, which is directly related to muscular contraction and subsequently to digging behavior, was also found to be affected by anthropogenic sound intensity. The findings suggest that sound may be perceived by bivalves as changes in the water particle motion and lead to the subsequent reactions detected in razor clams.

  13. Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats

    Yi-zong ZHAI


    Full Text Available Objective To explore the effects of percutaneous impulsive current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats, in order to investigate the effect of exercise-induced fatigue. Methods Seventy-two 8-week old male Wistar rats were randomly divided into 4 groups (18 each: control group (group A, fatigue group (group B, stimulation before fatigue group (group C and stimulation after fatigue group (group D. Exhaustion of animals in B, C and D groups were reproduced by prolonged swimming. Current stimulation (1024Hz, 10mA, current cycle 1sec for 20 minutes was given to the rats of group C before swimming, and to those in group D after exhaustion. At the weekend of 1st, 3rd and 5th week after modeling, the rats were sacrificed in batches from each group (6 each. The activities of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase were determined by spectrophotometry, and Bradfood protein quantification was employed to quantitate the protein in rats' hepatic mitochondria. Results No significant difference was found in swimming-exhaustion time among 3 groups at the first weekend (P>0.05, while the swimming-exhaustion time was significantly prolonged at the 3rd and 5th weekends in group D than in group B and C (P0.05, while the enzyme activities were obviously lower at the 3rd and 5th weekend in group B than that in groups A, C and D (P<0.05, and they were also lower in group C than that in group D (P<0.05. Conclusions Exercise-induced fatigue can lower the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase. Percutaneous pulsive current stimulating hepatic region of exercise-induced fatigued rats may improve the enzyme activity, reduce the concentration of free calcium and calcium overload in mitochondria, stimulate the oxidative phosphorylation, accelerate the rate of respiratory chain, promote exercise endurance and score, and

  14. Crystal Structure of the Vanadate-Inhibited Ca2+-ATPase

    Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand; Olesen, Claus; Andersen, Jens Peter; Møller, Jesper Vuust; Nissen, Poul


    catalytic site as a planar VO3− in complex with water and Mg2+ in a dephosphorylation transition-state-like conformation. Validating bound VO3− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization......Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound at the...

  15. 甘草黄酮对大强度耐力运动大鼠肾脏组织Ca2+、Mg2+-ATPase及Bax,Bc1-2蛋白表达的影响%Effect of High-Intensity Endurance Exercise on Ca2+,Mg2+-ATPase and Bax, Bcl-2 Protein Expression With Glycyrrhiza Flavonoids in rat Nephridial Tissue

    王东旭; 陈艳艳


    Objective To explore Glycyrrhiza Elavonoids on the rat nephridial tissue of Ca2+, Mg2+-ATPase and Bax, Bcl-2 protein expression with high-intensity endurance exercise. Methods The twenty-four healthy male rats were randomly divided into quiet groups, high-intensity exercise group and exercise plus Glycyrrhiza Elavonoids group, After 6 weeks of treadmill training, Using the box of reagent and immunity histochemistry examined the changing of Ca2+, Mg2+-ATPase and Bax, Bcl-2 protein expression on each groups . Results Compared with the quiet groups, the activity of Ca2+, Mg2+-ATPase both had significant droped (P<0.01), and the groups of plus drog had very difference increased than high-intendity exerxise groups (P<0.01); High-intensity endurance exercise group and exercise dosing rats AI apoptosis index increased in varying degrees;high-intensity exercise group (MOD) were very significant difference(P<0.01), exercise plus drug group Bac protein expression (MOD)were very significant difference (P<0.01); Exercise plus drug group Bcl-2 protein expression(MOD) with the high-intersity exercise group had significant difference(P<0.01), High-intensity exercise group and exercise plus drug group Bax/Bcl-2 ratio of distribution is significantly difference degrees of difference(P<0.05,P<0.01).%目的:探讨甘草黄酮对大强度耐力运动大鼠肾脏组织Ca2+、Mg2+-ATPase及Bax、Bcl-2表达的影响。方法:选取SD雄性健康大鼠24只,随机分为安静组、大强度运动组和运动加药组;采用跑台训练6周后取材,应用试剂盒和免疫组织化学法测检测各组大鼠肾脏组织Ca2+、Mg2+-TPase活性及Bax和Bcl-2表达的变化。结果:与安静对照组相比,大强度运动组和运动加药组肾脏组织Ca2+、Mg2+-TPase活性均呈非常显著性下降(P<0.01);其中运动加药组Ca2+、Mg2+-TPase活性均较大强度运动组具有非常显著差异性提高(P<0.01);大强度耐力运动组和运动加

  16. Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.

    Clausen, Johannes D; Bublitz, Maike; Arnou, Bertrand; Olesen, Claus; Andersen, Jens Peter; Møller, Jesper Vuust; Nissen, Poul


    Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase. PMID:27050689

  17. Relations between erythrocyte Ca2+ , Mg2+ levels and cell membrane ATPase activities in patients with hypertension in obese children%肥胖儿童红细胞钙镁水平及ATP酶活性与高血压关系的探讨

    朱树森; 符云峰; 卢振敏; 王素敏; 孙爱丽


    目的探讨细胞离子代谢紊乱在肥胖儿童高血压发病中的作用。方法测定125例(正常对照37例,正常血压肥胖34例,正常体重高血压21例,高血压肥胖33例)12~16岁中学生的红细胞膜ATP酶活性、血浆和红细胞胞浆Ca2+、Mg2+水平。结果正常血压肥胖组(ONT)儿童红细胞膜Na+-K+-ATP酶和Ca2+-ATP酶活性较正常对照组(NT)显著降低,正常体重高血压组(NOHT)和高血压肥胖组(OHT)两酶活性又显著低于ONT组。ONT组、NOHT组和OHT组红细胞胞浆Ca2+水平三组之间无显著差异,但均显著高于NT组。红细胞胞浆Mg2+,血浆Ca2+、Mg2+在NT组和ONT组之间无显著差异,在NOHT组和OHT组均显著降低。结论红细胞膜Na+-K+-ATP酶和Ca2+-ATP酶活性降低可能在肥胖儿童高血压的发病机制中有重要作用。%Objective To explore the roles of the metabolic disorders of cellular ions in pathogenesis of hypertension in obese children. Methods Erythrocyte membrane ATPase activities,plasma and cellular Ca2 + , Mg2 + levels were measured in 125(37 non-obese normotensives,34 obese normotensives,21 hypertensives and 33 obese hypertensives)middle school students aged 12~16 years. Results Erythrocyte membrane Na+ -K+-ATPase and Ca2+ -ATPase activities were significantly lower in obese children with normotension than those in non-obese normotensive children,and they were also significantly lower in two groups of hypertensive children than those in obese children with normotension. Erythrocyte Ca2+ in normotensive obese and two group of hypertensive children was significantly higher than that in non-obese normotensive children, but there were no significant differences between the three groups. There were no significant differences found in erythrocyte Mg2+ , plasma Ca2+ and Mg2+ between normotensive obese and normotensive non-obese children, but significant decreases were found in both groups of hypertensive children. Conclusion Decreased Na +-K+-ATPase

  18. The Influences of Mg2+ , Ca2+ and Mg2+/Ca2+ Ratio in Mixed Seawater on the Emergence Rate of Penaeus japonicus Postlarva

    臧维玲; 戴习林; 江敏; 姚庆祯; 蔡云龙; 罗春芳; 徐桂荣; 丁福江


    This paper reports the approprite ranges of Mg2+ , Ca2 + and their ratio Mg2 +/Ca2 + inmixed seawater for rearing of Penaeus japonicus larvae. The ranges for the above three indices are1150- 1450 mg/L, 360- 440 mg/L and 2.8 - 3.4, respectively. The proper sahnity range ofmixed seawater is 22.1 - 33.8 obtained by mixing estuarine water and concentrated seawater.

  19. Acid-base status determines the renal expression of Ca2+ and Mg2+ transport proteins.

    Nijenhuis, T.; Renkema, K.Y.R.; Hoenderop, J.G.J.; Bindels, R.J.M.


    Chronic metabolic acidosis results in renal Ca2+ and Mg2+ wasting, whereas chronic metabolic alkalosis is known to exert the reverse effects. It was hypothesized that these adaptations are mediated at least in part by the renal Ca2+ and Mg2+ transport proteins. The aim of this study, therefore, was

  20. Effect of Ca2+ and Mg2+ on CO2 Corrosion Behavior of Tube Steel

    ZHAO Guo-xian; LI Jian-ping; HAO Shi-ming; L(U) Xiang-hong; LI He-lin


    Effects of Ca2+ and Mg2+ on the CO2 corrosion behaviors of tube steel were studied in simulated oil-fieldenvironment. The influence of Ca2+ and Mg2+ on the corrosion rate and morphologies of corrosion product layerwas determined by scanning electron microscope and measuring mass loss. Potentiodynamic polarization and im-pedance spectroscopy were used to investigate the change of electrochemical characteristic parameters of corrosionproduct layer and corrosion dynamic process. The results show that with Ca2+ and Mg2+ in electrolyte, the mor-phologies and microstructures of corrosion product layer changed obviously, thus affecting the corrosion process.

  1. Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

    Alexander, R Todd; Beggs, Megan R; Zamani, Reza;


    Plasma Membrane Ca(2+)-ATPase's (PMCA) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial Ca(2+) channel, Trpv5. We therefore hypothesized that Pmca4 plays a significant...... distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a...... role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies...

  2. Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases

    Vandecaetsbeek, Ilse; Christensen, Søren Brøgger; Liu, Huizhen; Van Veldhoven, Paul P; Waelkens, Etienne; Eggermont, Jan; Raeymaekers, Luc; Møller, Jesper V; Nissen, Poul; Wuytack, Frank; Vangheluwe, Peter


    The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are...

  3. Ca2+ and Mg2+ binding induce conformational stability of Calfumirin-1 from Dictyostelium discoideum

    Bairagi C Mallick; Sa-Ouk Kang; Suman Jha


    The apo-Calfumirin-1 (CAF-1) binds to Ca2+ with high affinity and also to Mg2+ with high positive cooperativity. The thermal unfolding curves of wtCAF-1 monitored at neutral pH by CD spectroscopy are reversible and show different thermal stabilities in the absence or presence of Ca2+ and Mg2+ ions. Metalfree wtCAF-1 shows greater thermal stability than EF-IV mutant protein. We observed that GdnHCl-induced unfolding of apo-wtCAF-1 monitored by CD and fluorescence spectroscopies increases co-operative folding with approximately same C values. Binding of Ca2+ and Mg2+ ions to CAF-1 dramatically altered the fluorescence and CD spectra, indicating metal ion-induced conformational changes both in the wild-type and mutant proteins. The hydrophobic probe, ANS is used to observe alteration in surface hydrophobicity of the protein in different ligation states. In apo-wtCAF-1, the exposed hydrophobic surfaces are able to bind ANS which is in contrast to the unfolded or the metal ions ligated conformations. Isothermal titration calorimetry (ITC) resultsshow two possible independent binding sites of comparable affinity for the metal ions. However, their binding to the EF-IV E helix-loop-F helix mutant apo-protein happens with different affinities. The present study demonstrates that Ca2+ or Mg2+ binding plays a possible role in the conformational stability of the protein.

  4. Chelate titrations of Ca(2+) and Mg(2+) using microfluidic paper-based analytical devices.

    Karita, Shingo; Kaneta, Takashi


    We developed microfluidic paper-based analytical devices (μPADs) for the chelate titrations of Ca(2+) and Mg(2+) in natural water. The μPAD consisted of ten reaction zones and ten detection zones connected through narrow channels to a sample zone located at the center. Buffer solutions with a pH of 10 or 13 were applied to all surfaces of the channels and zones. Different amounts of ethylenediaminetetraacetic acid (EDTA) were added to the reaction zones and a consistent amount of a metal indicator (Eriochrome Black T or Calcon) was added to the detection zones. The total concentrations of Ca(2+) and Mg(2+) (total hardness) in the water were measured using a μPAD containing a buffer solution with a pH of 10, whereas only Ca(2+) was titrated using a μPAD prepared with a potassium hydroxide solution with a pH of 13. The μPADs permitted the determination of Ca(2+) and Mg(2+) in mineral water, river water, and seawater samples within only a few minutes using only the naked eye-no need of instruments. PMID:27181645

  5. The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and is essential for reproduction.

    Yamazaki, Daisuke; Miyata, Haruhiko; Funato, Yosuke; Fujihara, Yoshitaka; Ikawa, Masahito; Miki, Hiroaki


    Ca(2+) influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+) transporter, is required for Ca(2+) influx during capacitation. We find that Cnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a qualitative change in the mode of sperm motility during capacitation, is abrogated in Cnnm4-deficient sperm. In contrast, tyrosine phosphorylation of flagellar proteins, a hallmark of capacitation, is excessively augmented. These seemingly paradoxical phenotypes of Cnnm4-deficient sperm are very similar to those of sperm lacking a functional cation channel of sperm (CatSper) channel, which plays an essential role in Ca(2+) influx during sperm capacitation. Ca(2+) imaging analyses demonstrate that Ca(2+) influx is perturbed in Cnnm4-deficient sperm, and forced Ca(2+) entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germ-cell-specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca(2+) homeostasis. PMID:27006114

  6. Comparison of Ca2+ and Mg2+ enhancing aerobic granulation in SBR

    Two sequencing batch reactors (SBRs) were operated to investigate the effect of Ca2+ and Mg2+ augmentation on aerobic granulation. Reactor R1 was augmented with Ca2+ at 40 mg/L, while Mg2+ was added to the reactor R2 with 40 mg/L. Results showed that the reactor R1 had a faster granulation process compared with R2, and the mature granules in R1 showed better physical characteristics. However, the mature granules in R2 had the higher production yield of polysaccharides and proteins, and aerobic granules in R2 experienced a faster substrate biodegradation. Microbial and genetic characteristics in mature granules were analyzed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The results revealed that Mg2+ addition led to higher microbial diversity in mature granules. In addition, an uncultured bacterium (AB447697) was major specie in R1, and β-proteobacterium was dominant in R2. It can be concluded that Ca2+ had an important effect on physical properties of aerobic granules, while Mg2+ played a key role on biological properties during the sludge granulation.

  7. Mg(2+) differentially regulates two modes of mitochondrial Ca(2+) uptake in isolated cardiac mitochondria: implications for mitochondrial Ca(2+) sequestration.

    Blomeyer, Christoph A; Bazil, Jason N; Stowe, David F; Dash, Ranjan K; Camara, Amadou K S


    The manner in which mitochondria take up and store Ca(2+) remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca(2+) uptake and a complex Ca(2+) sequestration mechanism in mitochondria. But how Mg(2+) regulates these different modes of Ca(2+) uptake as well as mitochondrial Ca(2+) sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca(2+) by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca(2+) uptake, which was approximately 40 % of the slow uptake rate observed in protocol A. These two types of Ca(2+) uptake modes were differentially modulated by extra-matrix Mg(2+). That is, Mg(2+) markedly inhibited the slow mode of Ca(2+) uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg(2+) also inhibited Na(+)-dependent Ca(2+) extrusion. The general Ca(2+) binding properties of the mitochondrial Ca(2+) sequestration system were reaffirmed and shown to be independent of the mode of Ca(2+) uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg(2+) hindered Ca(2+) sequestration. Our results indicate that mitochondria exhibit different modes of Ca(2+) uptake depending on the nature of exposure to extra-matrix Ca(2+), which are differentially sensitive to Mg(2+). The implications of these findings in cardiomyocytes are discussed. PMID:26815005

  8. Thyroid hormone stimulation in vitro of red blood cell Ca2+-ATPase activity: interspecies variation.

    Davis, F B; Kite, J H; Davis, P J; Blas, S D


    In vitro susceptibility to thyroid hormone stimulation of membrane-associated Ca2+-ATPase activity has been examined in red blood cells from rat, rabbit, dog, monkey, and man. Monkey and human red cell Ca2+-ATPase activities responded comparably to 10(-10)M T4 or T3. Basal and thyroid hormone-stimulated Ca2+-ATPase activity in rabbit erythrocytes was four-fold higher than in primate red cells. Rat and dog red cell Ca2+-ATPase did not respond to iodothyronines in vitro. PMID:6459228

  9. Depressing effect of phenoxyl acetic acids on flotation of minerals containing Ca2+/Mg2+ gangues


    Phenoxyl acetic acids were applied to determine their depressing effect on minerals containing Ca2+/Mg2+ gangues. Calcite,mixture of calcite and fluorite, and nickel ore were used in the flotation. And the depression mechanism was studied by the determination of contact angle, zeta potential, adsorptive capacity of collector, and IR analysis as well. It is found that 0.1 mmol/L of phenoxyl acetic acid derived from pyrogallol or gallic acid exhibits strong depressing ability on calcite in almost zero yields at pH value of 9.8, and calcite can be depressed in the flotation of calcite/fluorite mixture for approximate 87% yield of fluorite. The flotation result of practical nickel ore containing serpentine indicates that these two depressants may also show better depression performance to serpentine than traditional depressants such as sodium fluosilicate and carboxylmethyl cellulose. Analysis for the depression mechanism reveals that there exists strong chemical interaction between the depressants and minerals.

  10. Isotopic fractionation of Mg 2+(aq), Ca 2+(aq), and Fe 2+(aq) with carbonate minerals

    Rustad, James R.; Casey, William H.; Yin, Qing-Zhu; Bylaska, Eric J.; Felmy, Andrew R.; Bogatko, Stuart A.; Jackson, Virgil E.; Dixon, David A.


    Density-functional electronic structure calculations are used to compute the equilibrium constants for 26Mg/ 24Mg and 44Ca/ 40Ca isotope exchange between carbonate minerals and uncomplexed divalent aquo ions. The most reliable calculations at the B3LYP/6-311++G(2d,2p) level predict equilibrium constants K, reported as 10 3ln ( K) at 25 °C, of -5.3, -1.1, and +1.2 for 26Mg/ 24Mg exchange between calcite (CaCO 3), magnesite (MgCO 3), and dolomite (Ca 0.5Mg 0.5CO 3), respectively, and Mg 2+(aq), with positive values indicating enrichment of the heavy isotope in the mineral phase. For 44Ca/ 40Ca exchange between calcite and Ca 2+(aq) at 25 °C, the calculations predict values of +1.5 for Ca 2+(aq) in 6-fold coordination and +4.1 for Ca 2+(aq) in 7-fold coordination. We find that the reduced partition function ratios can be reliably computed from systems as small as M(CO)610- and M(HO)62+ embedded in a set of fixed atoms representing the second-shell (and greater) coordination environment. We find that the aqueous cluster representing the aquo ion is much more sensitive to improvements in the basis set than the calculations on the mineral systems, and that fractionation factors should be computed using the best possible basis set for the aquo complex, even if the reduced partition function ratio calculated with the same basis set is not available for the mineral system. The new calculations show that the previous discrepancies between theory and experiment for Fe 3+-hematite and Fe 2+-siderite fractionations arise from an insufficiently accurate reduced partition function ratio for the Fe 3+(aq) and Fe 2+(aq) species.

  11. Isotopic Fractionation of Mg2+(aq), Ca2+(aq), and Fe2+(aq) with Carbonate Minerals

    Rustad, James R.; Casey, William H.; Yin, Qing-Zhu; Bylaska, Eric J.; Felmy, Andrew R.; Bogatko, Stuart A.; Jackson, Virgil E.; Dixon, David A.


    Density functional electronic structure calculations are used to compute the equilibrium constant (the isotope fractionation factor) for 26Mg/24Mg and 44Ca/40Ca isotope exchange between carbonate minerals and uncomplexed divalent aquo ions. The most reliable calculations at the B3LYP/6-311++G(2d,2p) level predict equilibrium constants K, reported as 103ln(K) at 25 °C, of -5.3, -1.1, and +1.1 for 26Mg/24Mg exchange between calcite (CaCO3), magnesite (MgCO3), and dolomite (Ca0.5Mg0.5CO3), respectively, and Mg2+(aq), with positive values indicating enrichment in the mineral phase. For 44Ca/40Ca exchange between calcite and Ca2+(aq), the calculations predict values of +1.5 for Ca2+(aq) in six-fold coordination and +4.1 for Ca2+(aq) in seven-fold coordination. We find that the reduced partition function ratios can be reliably computed from systems as small as M(CO3)610- and M2+(H2O)6 embedded in a set of fixed atoms representing the 2nd shell (and greater) coordination environment. We find that the aqueous cluster representing the aquo ion is much more sensitive to improvements in the basis set than the calculations on the mineral systems, and that fractionation factors should be computed using 2 the best possible basis set for the aquo complex, even if the reduced partition function ratio calculated with the same basis set is not available for the mineral system. The new calculations show that the previous discrepancies between theory and experiment for Fe3+-hematite and Fe2+-siderite fractionations arise from an insufficiently accurate reduced partition function ratio for the Fe3+(aq) and Fe2+(aq) species.

  12. Ca2+ and Na+ permeability of high-threshold Ca2+ channels and their voltage-dependent block by Mg2+ ions in chick sensory neurones.

    Carbone, E; Lux, H D; Carabelli, V; Aicardi, G; Zucker, H


    1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] ( 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990). PMID:9350613

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

    Godiksen, Helene; Jessen, Flemming


    Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes in...

  14. Total soil electrical conductivity and critical soil K+ to Ca2+ and Mg2+ ratio for potato crops

    Reis Jr. Roberto Anjos; Fontes Paulo Cezar Rezende; Neves Júlio Cesar Lima; Santos Nerilson Terra


    Soil K+ to Ca2+ and Mg2+ ratio as well as the total salinity were evaluated in response to potassium fertilizer application onto potato. Potassium was applied at six different rates (0, 60, 120, 240, 480 and 960 kg ha-1 of K2O), as K2SO4, and was placed during planting time in the furrow. Soil from the 0-200 mm layer was collected in the furrow, 20 and 48 days after plant emergence (DAE) to evaluate soil pH, K+, Ca2+ and Mg2+ contents and the total electrical conductivity (EC). A factorial de...

  15. Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

    Lawrence, W D; Davis, P J; Blas, S D


    The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up ...

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

    Sarcoplasmic reticulum Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca2+-ATPase occurred within a few hours in the presence of ≤ 50 μM Ca2+. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high 45Ca2+ concentration (500 μM), monomeric Ca2+-ATPase was stable for several house. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca2+-ATPase was found to be 105-106 M-1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca2+-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca2+ and 48V vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. The results suggest that formation of Ca2+-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit

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

    Godiksen, Helene; Jessen, Flemming


    the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca2+-dependence of SR Ca2+-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo, salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2......+- ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degreesC in salmon and rainbow trout, respectively. Ca2+-dependence in the four different fish species investigated was very similar with half maximal activation (K-0.5) between 0.2 and 0.6 muM and half maximal inhibition (I-0...

  18. Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

    Li, Kai; Zhao, Lihong; Geng, Guangrui; Ma, Liqin; Dong, Shishan; Xu, Tong; Wang, Jianlin; Wang, Huiyu; Tian, Yong; Qiao, Jian


    The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers. PMID:20728193

  19. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    Drachmann, Nikolaj Düring


    a from pig hearts is presented. The purified protein was used for X-ray crystallographic studies aiming at determining the three dimensional structure of the SERCA2a isoform in a Ca2+-free conformation. Crystals of the Ca2+ free state of SERCA2a stabilised by the inhibitor cyclopiazonic acid was...... obtained and a dataset was collected scaling to 3.26 Å resolution, allowing a preliminary structural analysis. The overall crystal structure is very similar to SERCA1a. Additionally, co-crystallisation studies have been initiated of SERCA2a and recombinantly expressed Phospholamban. Besides the above...... resolution of the proof-of-concept Ca2+ bound crystal form, indicated that the information content of SFX data is higher than synchrotron data, and ligands and ions can be detected with low redundant data. The data of the E2 stabilised form was processed to 5 Å resolution, and it was possible to extract...

  20. Mg(2+)/Ca(2+) promotes the adhesion of marine bacteria and algae and enhances following biofilm formation in artificial seawater.

    He, Xiaoyan; Wang, Jinpeng; Abdoli, Leila; Li, Hua


    Adhesion of microorganisms in the marine environment is essential for initiation and following development of biofouling. A variety of factors play roles in regulating the adhesion. Here we report the influence of Ca(2+) and Mg(2+) in artificial seawater on attachment and colonization of Bacillus sp., Chlorella and Phaeodactylum tricornutum on silicon wafer. Extra addition of the typical divalent cations in culturing solution gives rise to significantly enhanced adhesion of the microorganisms. Mg(2+) and Ca(2+) affect the adhesion of Bacillus sp. presumably by regulating aggregation and formation of extracellular polymeric substances (EPS). The ions alter quantity and types of the proteins in EPS, in turn affecting subsequent adhesion. However, it is noted that Mg(2+) promotes adhesion of Chlorella likely by regulating EPS formation and polysaccharide synthesis. Ca(2+) plays an important role in protein expression to enhance the adhesion of Chlorella. For Phaeodactylum tricornutum, Ca(2+) expedites protein synthesis for enhanced adhesion. The results shed some light on effective ways of utilizing divalent cations to mediate formation of biofilms on the marine structures for desired performances. PMID:27362920

  1. Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1.

    Balog, E M; Fruen, B R; Shomer, N H; Louis, C F


    The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-si...

  2. Photoproducts of tetracycline and oxytetracycline involving self-sensitized oxidation in aqueous solutions: Effects of Ca2+ and Mg2+

    Yong Chen; Hua Li; Zongping Wang; Tao Tao; Chun Hu


    Tetracyclines constitute one of the most important antibiotic families and represent a classic example of phototoxicity.The photoproducts of tetracyclines and their parent compounds have potentially adverse effects on natural ecosystem.In this study,the self-sensitized oxidation products of tetracycline (TC) and oxytetracycline (OTC) were determined and the effects of Ca2+ and Mg2+on self-sensitized degradation were investigated.The Ca2+ and Mg2+ in the natural water sample accounted for enhancement (pH 7.3)and inhibition (pH 9.0) of photodegradation of TC and OTC due to the formation of metal-ions complexes.The formation of Mg2+ complexes was unfavorable for the photodegradation of the tetracyclines at both pH values.In contrast,the Ca2+ complexes facilitated the attack of singlet oxygen (1O2) arising from self-sensitization at pH 7.3 and enhanced TC photodegradation.For the first time,selfsensitized oxidation products of TC and OTC were verified by quenching experiments and detected by LC/ESI-DAD-MS.The products had a nominal mass 14 Da higher than the parent drugs (designated M+14),which resulted from the 1O2 attack of the dimethylamino group on the C-4 atom of the tetracyclines.The presence of Ca2+ and Mg2+ also affected the generation of M+14 due to the formation of metal-ions complexes with TC and OTC.The findings suggest that the metal-ion complexation has significant impact on the selfsensitized oxidation processes and the photoproducts of tetracyclines.

  3. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde


    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  4. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed 45Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of 45Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis

  5. Penentuan Kesadahan Ca 2+ dan Mg 2+ Pada Air Baku dan Air Resevoir Di PDAM Tirtanadi Instalasi Delituamedan

    Harahap, Mahyusar


    Has conducted analysis on the raw water hardness and water reservoir in PDAM Tirtanadi Installation Delitua Medan. Mainly caused by water hardness ions - ions Ca2 + and Mg2+ , magnesium and calcium hardness is determined by titration method using Na2EDTA and indicators EBT to total hardness and mureksid indicator for the level of calcium. Magnesium hardness value is determined by the reduction in volume (ml) Na2EDTA titration the total hardness of CaCO3 with the volume (ml) Na2EDTA titration ...

  6. All Ca(2+)-binding loops of light-sensitive ctenophore photoprotein berovin bind magnesium ions: The spatial structure of Mg(2+)-loaded apo-berovin.

    Burakova, Ludmila P; Natashin, Pavel V; Malikova, Natalia P; Niu, Fengfeng; Pu, Mengchen; Vysotski, Eugene S; Liu, Zhi-Jie


    Light-sensitive photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola. Berovin is functionally identical to the well-studied Ca(2+)-regulated photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca(2+)-binding sites and consequently belongs to a large family of the EF-hand Ca(2+)-binding proteins. Here we report the spatial structure of apo-berovin with bound Mg(2+) determined at 1.75Å. The magnesium ion is found in each functional EF-hand loop of a photoprotein and coordinated by oxygen atoms donated by the side-chain groups of aspartate, carbonyl groups of the peptide backbone, or hydroxyl group of serine with characteristic oxygen-Mg(2+) distances. As oxygen supplied by the side-chain of the twelfth residue of all Ca(2+)-binding loops participates in the magnesium ion coordination, it was suggested that Ca(2+)-binding loops of berovin belong to the mixed Ca(2+)/Mg(2+) rather than Ca(2+)-specific type. In addition, we report an effect of physiological concentration of Mg(2+) on bioluminescence of berovin (sensitivity to Ca(2+), rapid-mixed kinetics, light-sensitivity, thermostability, and apo-berovin conversion into active protein). The different impact of physiological concentration of Mg(2+) on berovin bioluminescence as compared to hydromedusan photoproteins was attributed to different affinities of the Ca(2+)-binding sites of these photoproteins to Mg(2+). PMID:26690016

  7. A tomato ER-type Ca2+-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese

    Johnson, Neil A.; Liu, F; Weeks, P. D.;


    Recombinant Ca(2+)-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We...... investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca(2+)-ATPases. Enzyme function was confirmed by the ability of each Ca(2+)-ATPase to rescue K616 growth on EGTA-containing agar and...

  8. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))


    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  9. Increased calcium deposits and decreased Ca2+-ATPase in right ventricular myocardium of ascitic broiler chickens.

    Li, K; Qiao, J; Zhao, L; Dong, S; Ou, D; Wang, J; Wang, H; Xu, T


    Right ventricular hypertrophy and failure is an important step in the development of ascites syndrome (AS) in broiler chickens. Cytoplasmic calcium concentration is a major regulator of cardiac contractile function and various physiological processes in cardiac muscle cells. The purpose of this study was to measure the right ventricular pressure and investigate the precise ultrastructural location of Ca(2+) and Ca(2+)-ATPase in the right ventricular myocardium of chickens with AS induced by low ambient temperature. The results showed that the right ventricular diastolic pressure of ascitic broilers was significantly higher than that of control broilers (P ascitic broilers was significantly lower than that of the controls (P ascitic broilers, whereas in the age-matched control broilers, calcium deposits were much less. The Ca(2+)-ATPase reactive products were obviously found on the sarcoplasmic reticulum and mitochondrial membrane of the control right ventricular myocardium, but rarely observed in the ascitic broilers. The data suggest that in ascitic broilers there is the right ventricular diastolic dysfunction, in which the overload of intracellular calcium and the decreased Ca(2+)-ATPase activity might be the important factors. PMID:17054481

  10. Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

    Reis, M; Farage, M; de Souza, A C; de Meis, L


    The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production. PMID:11544263

  11. Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

    The GTP-driven component of Ca2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca2+-translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45Ca2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca2+-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45Ca2+-uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45Ca2+ uptake represents the capacity of the plasma membrane Ca2+-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-32P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca2+-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca2+-ATPases present in animal cells

  12. Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ Binding

    Sillen, Alain; Verheyden, Stefan; Delfosse, Lotte; Braem, Tania; Robben, Johan; Volckaert, Guido; Engelborghs, Yves


    The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca2+ or Mg2+. Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca2+ or Mg2+. Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of th...

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

    Naoko Iguchi

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

  14. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Prabhanshu Kumar


    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  15. Plasma membrane Ca2+-ATPases in the nervous system during development and ageing

    Ana; M; Mata; M; Rosario; Sepulveda


    Calcium signaling is used by neurons to control a variety of functions,including cellular differentiation,synaptic maturation,neurotransmitter release,intracellular signaling and cell death.This review focuses on one of the most important Ca2+regulators in the cell,the plasma membrane Ca2+-ATPase(PMCA),which has a high affinity for Ca2+and is widely expressed in brain.The ontogeny of PMCA isoforms,linked to specific requirements of Ca2+ during development of different brain areas,is addressed, as well as their function in the adult tissue.This is based on the high diversity of variants in the PMCA family in brain,which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely,alterations in the activity of PMCAs could lead to changes in Ca2+homeostasis and,consequently,to neural dysfunction.The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed.

  16. Propofol regulates Ca2+, Mg2+, Cu2+ and Zn2+ balance in the spinal cord after ischemia/reperfusion injury

    Shuzhou Yin; Qijing Yu; Ji Hu; Jie Yang; Juan Chen


    This study assessed concentrations of Ca2+, Mg2+, Cu2+ and Zn2+ in blood serum and spinal cord tissues, as well as the possible mechanisms by which propofol may protect spinal cord tissues during ischemia/reperfusion injury. With prolonged duration of ischemia/reperfusion injury, serum Ca2+ and Cu2+ concentrations gradually increased, but Mg 2+ and Zn2+ concentrations gradually decreased. Seven days after spinal cord injury, changes in Ca2+, Mg2+, Cu2+ and Zn2+ concentrations were significant. After 7 days of reperfusion, changes in the concentrations of Ca2+, Mg2+, Cu2+ and Zn2+ in spinal cord homogenates were consistent with those in the serum. After propofol treatment, no significant changes in Ca2+, Mg2+, Cu2+ and Zn2+ concentrations in serum and spinal cord homogenates were noted during ischemia/reperfusion injury. These findings suggest that propofol exerts protective effects against spinal cord injury by stabilizing or recovering metal ion balance in ischemic regions.

  17. Photoluminescence of Vanadate Garnet Ca2NaMg(2-x)V3O12:xEu3+ Phosphors Synthesized by Solution Combustion Method.

    Kim, H; Kim, J; Lim, S; Park, K


    In this study, a series of nano-sized Ca2NaMg(2-x)V3O12:xEu3+ (0.06 phosphors is synthesized by solution combustion method. The microstructure and photoluminescence properties of the Ca2NaMg(2-x)V3O12:xEu3+ phosphors are studied in accordance with the Eu3+ content. Annealed Ca2NaMg(2-x)V3O12:xEu3+ phosphors form a single phase with the cubic garnet structure and Ia3d space group. The emission from (VO4)3- in the Ca2NaMg(2-x)V3O12:xEu3+ phosphors is almost completely quenched due to the efficient energy transfer from the (VO4)3- to the Eu3+ ions. The emission intensity increases sharply with the increased Eu3+ content, reaching a maximum value at x = 0.15, and then decreases with further Eu3+ content. Ca2NaMg1.85V3O12:0.15Eu3+ shows great potential as a red phosphor for white light-emitting diodes. PMID:27433680

  18. Plasma membrane Ca2+-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

    Asma; Zaidi


    The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

  19. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Ju-Hyun Lee


    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  20. Changes of mitochondrial structure, ATPase and Ca2+ concentration in spermatogenic cells of mouse testes induced by low dose radiation

    Objective: To observe the ultrastructure, ATPase activity and Ca2+ concentration ([Ca2+]i) of mitochondria in the sperematogenic cells of mouse testes 3-24 h after low dose radiation with 0.025-0.200 Gy X-rays, and illuminate the effects of mitochondrion structure and relative biological function on apoptosis. Methods: The ultrastructure changes of mitochondria in the spermatogenic cells were observed with transmission electron microscope; the ATPase activity was measured with protein enzymic method; [Ca2+]i was measured indirectly by flow cytometry with Fluo-3 probes. Results: The mitochondria swelled and vacuolizated, and their cristae were broken in the spermatogonia and spermatocytes 12 h after irradiation, and their nuclei were karyopyknosis, the acrosomal vesicle structure was ambiguity, the membrane structure was unclear, and the mitochondria in spermatids were vacuolization. The activities of Na+-K+-ATPase in mouse testis tissue 12 h after irradiated with 0.025-0.200 Gy decreased compared with those with 0 Gy, the Na+-K+-ATPase activities of the cells irradiated with 0.05-0.200 Gy decreased significantly compared with those with 0 Gy (P2+-ATPase of the cells irradiated with 0.025-0.200 Gy decreased significantly compared with those with 0 Gy (P2+]i in mouse testis spermatogenic cells had similar dose-response relationship, [Ca2+]i after irradiated with 0.075 Gy decreased compared with those with 0 Gy (P+-K+-ATPase in mouse testis tissues decreased obviously compared with those at 0 h (P2+-ATPase in mouse testis tissues increased slightly at 3 h, then decreased at 6-24 h compared with those at 0 h (P2+]i in mouse testis spermatogenic cells had similar time course-response relationship, [Ca2+]i at 12 h decreased significantly compared with at 0 h (P2+]i induced by low dose radiation. (authors)

  1. Contribution of plasma membrane Ca2+ ATPase to cerebellar synapse function

    Helena; Huang; Raghavendra; Y; Nagaraja; Molly; L; Garside; Walther; Akemann; Thomas; Knpfel; Ruth; M; Empson


    The cerebellum expresses one of the highest levels of the plasma membrane Ca2+ATPase,isoform 2 in the mammalian brain.This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex;i.e. the Purkinje neurons(PNs) .Here we review recent evidence,including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2(PMCA2) knockout mouse,to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour.These studies have also revealed that deletionof PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development,they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

  2. 海水对兔眼晶状体的Na,K-ATP酶及Ca2,Mg2-ATP酶功能的影响%Experimental Study on the Effects of Seawater on Na+,K+-ATP ase and Ca2+,Mg2+-ATP ase in Lens of Rabbits

    徐绍娟; 彭秀军; 魏翠荣; 竹颖


    Objective:To investigate the changes of Na+,K+-ATP ase and Ca2+,Mg2+-ATP ase in lens of rabbits with seawater injected into anterior chambers.Methods:After 0.2 ml seawater were injected into anterior chambers,the rabbit’s eye balls were enucleated respectively in deferent time to examine the activity of Na+,K+-ATP ase and Ca2+,Mg2+-ATP ase in lens by biochemistry method.Results:The activity of Na+,K+-ATP ase and Ca2+,Mg2+-ATP ase in lens of seawater group decreased significantly at first,then increased and recovered partly or completely at last.No significant difference of the activity of two kinds of enzyme in lens was found between control group and normal group.Conclusion:The activity of Na+,K+-ATP ase and Ca2+,Mg2+-ATP ase in rabbit lens are damaged in deferent degree with seawater injected into anterior chamber.%  目的:观察海水对兔眼晶状体Na+,K+-ATP酶及Ca2+,Mg2+-ATP酶功能的影响.方法:在兔眼前房内注入0.2 ml海水后不同时间摘除眼球,分别测定其Na+,K+-ATP酶及Ca2+,Mg2+-ATP酶活性.结果:海水组兔眼晶状体两酶活性均先降低,再增高,最终完全或部分恢复正常;而对照组两酶的活性与正常组比较没有显著差别.结论:海水可使晶状体的Na+,K+-ATP酶及Ca2+,Mg2+-ATP酶的活性不同程度受损.

  3. Probabilidade de resposta da cana-de-açúcar à adubação potássica em razão da relação K+ (Ca2++Mg2+ -0,5 do solo Probability of sugarcane response to potassium fertilizer as a function of soil K+ (Ca2++Mg2+ -0,5 ratio

    Roberto dos Anjos Reis Junior


    Full Text Available O objetivo deste trabalho foi avaliar a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica, em razão da relação K+ (Ca2++Mg2+ -0,5 no solo. Foram compilados dados de 106 experimentos de adubação potássica na cana-de-açúcar. Em cada experimento foi registrado o ciclo de cultivo (cana-planta ou cana-soca, os teores de K, Ca e Mg do solo antes da adubação potássica, a relação K+ (Ca2++Mg2+ -0,5, e se houve, ou não, resposta estatisticamente significativa da produção à adubação potássica. Foi utilizado o método estatístico de regressão logística, efetuado pelo procedimento CATMOD do Statistical Analysis System. A característica ciclo de cultivo foi eliminada do modelo, pois esta se apresentou como não-significativa no ajuste estatístico. A relação K+ (Ca2++Mg2+ -0,5 do solo influenciou a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica. À medida que a relação K+ (Ca2++Mg2+ -0,5 aumentou, a probabilidade de resposta da produção de cana-de-açúcar à adubação potássica diminuiu. A relação K+ (Ca2++Mg2+ -0,5 no solo foi classificada em baixa (0,3349. A relação K+ (Ca2++Mg2+ -0,5 no solo deve ser usada como mais um critério para orientar a adubação potássica na cultura da cana-de-açúcar.This study was carried out to evaluate the effect of soil K+ (Ca2++Mg2+ -0.5 ratio on sugarcane yield response probability to potassium fertilizer. Results of 106 experiments of potassium fertilizer on sugarcane and their soil exchangeable K+, Ca2+ and Mg2+ were studied, evaluating the K+ (Ca2++Mg2+ -0.5 ratio in each experiment. The statistical method of logistic regression was used, carried out through CATMOD procedure of Statistical Analysis System. There was no difference of behavior of this ratio between plant cane and ratoons; therefore this characteristic was not significant during the adjustment of the statistical model. Soil K+ (Ca2++Mg2+ -0

  4. Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+-ATPase.

    Huang, Yongqi; Li, Huifang; Bu, Yuxiang


    Calcium ATPase is a member of the P-type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X-ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca2+ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca2+ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca2+ ion binds to site I, and then, the second Ca2+ ion approaches. The interactions between the second Ca2+ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca2+. Because of the electrostatic repulsion of the second Ca2+ and the electrostatic attraction of site II, the first bound Ca2+ shifts from site I to site II. Concertedly, the second Ca2+ binds to site I, forming a binding state with two Ca2+ ions, one at site I and the other at site II. Both of Glu908 and Asp800 coordinate with the two Ca2+ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. PMID:19242958

  5. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase

    Thastrup, Ole; Cullen, P J; Drøbak, B K;


    Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca...

  6. The Relationship Between Senescence and Ca2+-ATPase Activity of Microsomal Membrane and Lipid Peroxidation in Harvested Peach Fruit

    GUAN Jun-feng; FAN Xiu-cai; DOU Shi-juan; ZHANG Ji-shu; LI Guang-min


    Peach fruit easily soften and have a short storage time at normal temperature. In this study, peach fruit (Prunus persica sieb et Zucc cv. Yingqing) were picked and stored at 25 and 4℃ to investigate the senescence in correlation with Ca2+- ATPase activity of microsomal membrane and lipid peroxidation during ripening and senescence. In comparison with that stored at 25℃, the fruit stored at 4℃ exhibited a higher flesh firmness, lower respiration rate, and generated the late bigger peak value of Ca2+-ATPase activity as well as maintained the higher activity of the enzyme. Meanwhile, the lower levels of super oxygen radical (O2-) production and content of malondialdehyde (MDA), a product of membrane lipid peroxidation were observed. Sodium orthovanadate (SO) and erythrosin B (EB), as Ca2+-ATPase inhibitors, could stimulate the respiration rate. The results suggested that the slower senescence rate of peach fruit was closely related to the higher peak value and longer duration of Ca2+-ATPase activity in microsomal membrane, with the slighter membrane lipid peroxidation and lower O2(-) production rate.

  7. A sulphonated carbon dot-chitosan hybrid hydrogel nanocomposite as an efficient ion-exchange film for Ca2+ and Mg2+ removal

    Baruah, Upama; Konwar, Achyut; Chowdhury, Devasish


    We have developed a hybrid hydrogel nanocomposite film via conjugation of oxidised carbon dots synthesized from 11-mercaptoundecanoic acid with chitosan. The potential applicability of the film was then successfully tested for the removal of Ca2+ and Mg2+ ions from solution.We have developed a hybrid hydrogel nanocomposite film via conjugation of oxidised carbon dots synthesized from 11-mercaptoundecanoic acid with chitosan. The potential applicability of the film was then successfully tested for the removal of Ca2+ and Mg2+ ions from solution. Electronic supplementary information (ESI) available: The ESI includes the detailed synthesis and characterization of carbon dots both before and after oxidation and of the carbon dot-chitosan nanocomposite films viz. DLS, SEM, UV-visible, FTIR, PL spectroscopy and TGA. See DOI: 10.1039/c6nr01129b

  8. Role of platelet plasma membrane Ca2+-ATPase in health and disease

    William; L; Dean


    Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

  9. Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3.

    Tauber, Philipp; Aichinger, B; Christ, C; Stindl, J; Rhayem, Y; Beuschlein, F; Warth, R; Bandulik, S


    Adrenal aldosterone-producing adenomas (APAs) are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. These stimuli lead to a depolarization of the plasma membrane and, as a consequence, an increase of intracellular Ca(2+). Mutations of the plasma membrane Ca(2+)-ATPase ATP2B3 have been found in APAs with a prevalence of 0.6%-3.1%. Here, we investigated the effects of the APA-associated ATP2B3(Leu425_Val426del) mutation in adrenocortical NCI-H295R and human embryonic kidney (HEK-293) cells. Ca(2+) measurements revealed a higher basal Ca(2+) level in cells expressing the mutant ATP2B3. This rise in intracellular Ca(2+) was even more pronounced under conditions with high extracellular Ca(2+) pointing to an increased Ca(2+) influx associated with the mutated protein. Furthermore, cells with the mutant ATP2B3 appeared to have a reduced capacity to export Ca(2+) suggesting a loss of the physiological pump function. Surprisingly, expression of the mutant ATP2B3 caused a Na(+)-dependent inward current that strongly depolarized the plasma membrane and compromised the cytosolic cation composition. In parallel to these findings, mRNA expression of the cytochrome P450, family 11, subfamily B, polypeptide 2 (aldosterone synthase) was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3. In summary, the APA-associated ATP2B3(Leu425_Val426del) mutant promotes aldosterone production by at least 2 different mechanisms: 1) a reduced Ca(2+) export due to the loss of the physiological pump function; and 2) an increased Ca(2+) influx due to opening of depolarization-activated Ca(2+) channels as well as a possible Ca(2+) leak through the mutated pump. PMID:27035656

  10. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Nedukha, Olena


    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  11. Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

    Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey


    Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. PMID:24723394

  12. Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

    Kazi Md Kamrul Huda

    Full Text Available BACKGROUND: Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. RESULTS: The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. CONCLUSIONS: The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the

  13. Engineering a prototypic P-type ATPase Listeria Monocytogenes Ca(2+)-ATPase 1 for single-molecule FRET studies

    Dyla, Mateusz; Andersen, Jacob; Kjaergaard, Magnus;


    Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrol...

  14. Effect of ionizing radiation on catalytic properties of Ca2+-ATP-ase from sarcoplasmic reticulum of skeletal muscle

    It was studied kinetic and thermodynamic characteristics of Ca2+-ATP-ase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with disbalance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

  15. Effect of ionizing radiation on catalytic properties of Ca2+-ATPase from sarcoplasmic reticulum of skeletal muscle

    It was studied kinetic and thermodynamic characteristics of Ca2+-ATPase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with dis balance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

  16. Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding.

    Sillen, Alain; Verheyden, Stefan; Delfosse, Lotte; Braem, Tania; Robben, Johan; Volckaert, Guido; Engelborghs, Yves


    The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca(2+) or Mg(2+). Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca(2+) or Mg(2+). Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of these mutants show a limited decrease of the affinity for calcium, but no alterations of the cooperativity. Upon adding calcium, Trp170 shows a strong fluorescence increase, Trp57 an extensive fluorescence decrease, and Trp4 shows no fluorescence change. Therefore mutant W4F/W170F is ideally suited to analyze the fluorescence titrations and to study the binding mechanism. Mutations of the calcium ligands at the z-position in the three binding sites show no effect at site I and a total loss of cooperativity at sites III and IV. The quenching of Trp57 upon calcium binding is dependent on the presence of arginine R25, but this residue is not just a simple dynamic quencher. The role of the salt bridge R25-D58 is also investigated. PMID:12944301

  17. Affinity of Smectite and Divalent Metal Ions (Mg2+, Ca2+, Cu2+) with L-leucine: An Experimental and Theoretical Approach Relevant to Astrobiology

    Pandey, Pramod; Pant, Chandra Kala; Gururani, Kavita; Arora, Priyanka; Pandey, Neetu; Bhatt, Preeti; Sharma, Yogesh; Negi, Jagmohan Singh; Mehata, Mohan Singh


    Earth is the only known planet bestowed with life. Several attempts have been made to explore the pathways of the origin of life on planet Earth. The search for the chemistry which gave rise to life has given answers related to the formation of biomonomers, and their adsorption on solid surfaces has gained much attention for the catalysis and stabilization processes related to the abiotic chemical evolution of the complex molecules of life. In this communication, surface interactions of L-leucine (Leu) on smectite (SMT) group of clay (viz. bentonite and montmorillonite) and their divalent metal ion (Mg2+, Ca2+ and Cu2+) incorporated on SMT has been studied to find the optimal conditions of time, pH, and concentration at ambient temperature (298 K). The progress of adsorption was followed spectrophotometrically and further characterized by FTIR, SEM/EDS and XRD. Leu, a neutral/non polar amino acid, was found to have more affinity in its zwitterionic form towards Cu2+- exchanged SMT and minimal affinity for Mg2+- exchanged SMT. The vibrational frequency shifts of —NH3 + and —COO- favor Van der Waal's forces during the course of surface interaction. Quantum calculations using density functional theory (DFT) have been applied to investigate the absolute value of metal ion affinities of Leu (Leu—M2+ complex, M = Mg2+, Ca2+, Cu2+) with the help of their physico-chemical parameters. The hydration effect on the relative stability and geometry of the individual species of Leu—M2+ × (H2O)n, ( n =2 and 4) has also been evaluated within the supermolecule approach. Evidence gathered from investigations of surface interactions, divalent metal ions affinities and hydration effects with biomolecules may be important for better understanding of chemical evolution, the stabilization of biomolecules on solid surfaces and biomolecular-metal interactions. These results may have implications for understanding the origin of life and the preservation of biomarkers.

  18. Aluminum resistance in wheat involves maintenance of leaf Ca(2+) and Mg(2+) content, decreased lipid peroxidation and Al accumulation, and low photosystem II excitation pressure.

    Moustaka, Julietta; Ouzounidou, Georgia; Bayçu, Gülriz; Moustakas, Michael


    The phytotoxic aluminum species (Al(3+)) is considered as the primary factor limiting crop productivity in over 40 % of world's arable land that is acidic. We evaluated the responses of two wheat cultivars (Triticum aestivum L.) with differential Al resistance, cv. Yecora E (Al-resistant) and cv. Dio (Al-sensitive), exposed to 0, 37, 74 and 148 μM Al for 14 days in hydroponic culture at pH 4.5. With increasing Al concentration, leaf Ca(2+) and Mg(2+) content decreased, as well as the effective quantum yield of photosystem II (PSII) photochemistry (Φ PSII ), while a gradual increase in leaf membrane lipid peroxidation, Al accumulation, photoinhibition (estimated as F v /F m ), and PSII excitation pressure (1 - q p ) occurred. However, the Al-resistant cultivar with lower Al accumulation, retained larger concentrations of Ca(2+) and Mg(2+) in the leaves and kept a larger fraction of the PSII reaction centres (RCs) in an open configuration, i.e. a higher ratio of oxidized to reduced quinone A (QA), than plants of the Al-sensitive cultivar. Four times higher Al concentration in the nutrient solution was required for Al-resistant plants (148 μM Al) than for Al-sensitive (37 μM Al), in order to establish the same closed RCs. Yet, the decline in photosynthetic efficiency in the cultivar Dio was not only due to closure of PSII RCs but also to a decrease in the quantum yield of the open RCs. We suggest that Al(3+) toxicity may be mediated by nutrient deficiency and oxidative stress, and that Al-resistance of the wheat cultivar Yecora E, may be due at least partially, from the decreased Al accumulation that resulted to decreased reactive oxygen species (ROS) formation. However, under equal internal Al accumulation (exposure Al concentration: Dio 74 μM, Yecora E 148 μM) that resulted to the same oxidative stress, the reduced PSII excitation pressure and the better PSII functioning of the Al-resistant cultivar was probably due to the larger concentrations of Ca

  19. Dynamic Changes of Ca~(2+) and Ca~(2+)-ATPase in Sieve Elements in the Developing Caryopsis of Triticum aestivum L.%Ca~(2+)和Ca~(2+)-ATPase在小麦颖果筛分子分化中的动态变化

    李继伟; 邓祥宜; 周竹青; 王利凯; 阳超男; 樊海燕


    [Objective] Previous study revealed that sieve elements (SEs) in the developing caryopsis of Triticum aestivum L.underwent a unique type of programmed cell death (PCD).In this paper,the dynamic changes and the roles of Ca~(2+) and Ca~(2+)-ATPase in SEs during the PCD were studied.[Method] The ultrastructural aspects of phloem cells in wheat caryopsis were examined by transmission electron microscopy (TEM).Using specific fluorescence staining and potassium pyroantimonate precipitation method,Ca~(2+) was localized at histological and sub-cellular levels in SEs in the developing wheat caryopsis.TEM and lead nitrate were used to locate Ca~(2+)-ATPase in SEs.[Result] TEM studies showed that the cell walls of SEs thickened at the beginning of differentiation,and then became thinner and smoother.Fluorescence staining showed that the fluorescence due to Ca~(2+) appeared in cell walls of SEs from 6 to 10 d after flowering.The fluorescence due to Ca~(2+) in cell walls of SEs was most notable on 9 d after flowering and disappeared on 14 d after flowering.Sub-celluar localization of Ca~(2+) showed that Ca~(2+) was localized on plasma membrane and in nuclei from 1 to 2 d after flowering.On 4 d after flowering,Ca2+ was localized in cytoplasm and mitochondria of SEs.From 5 to 8 d after flowering,Ca~(2+) was transported to the cell walls of SEs and no Ca~(2+) precipitates were observed in mitochondria.From 10 to 18 d after flowering,Ca~(2+) was transported into the cytoplasm again from cell walls and no Ca~(2+) precipitates were observed on 20 d after flowering.In intermediary cells (ICs),Ca~(2+) precipitates were observed from 1 to 18 d after flowering,and Ca~(2+) mainly distributed on intine and tonoplast.The activity of Ca~(2+)-ATPase changed obviously during the SEs differentiation.There was lowest activity of Ca~(2+)-ATPase on 3 d after flowering in SEs.High levels of Ca~(2+)-ATPase activity were found from 4 to 14 d after flowering in SEs,and the enzyme was mainly localized

  20. Pycnogenol® and Ginkgo biloba extract: effect on peroxynitrite-oxidized sarcoplasmic reticulum Ca2+-ATPase

    Žižková, Petronela; Viskupičová, Jana; Horáková, L'ubica


    The effect of two natural standardized plant extracts, Pycnogenol® and EGb 761, on sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and posttranslational modifications induced by peroxynitrite was investigated to assess their possible protective role. EGb 761 was found to have a protective effect on SERCA activity in the concentration range of 5–40 µg/ml. On the other hand, Pycnogenol® caused a decrease of SERCA activity at concentrations of 25 µg/ml. EGb 761 did not prevent protein carbon...

  1. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone;


    of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...... crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin....

  2. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg; Nissen, Poul


    presynaptic and postsynaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium......Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for...

  3. 女贞子提取物对大鼠不同组织Ca2+-ATPase活性的影响%Effects of Ligustrum lucidum Extracts on Ca2+-ATPase Activity in Different Tissues of Rat

    马云慧; 熊正英


    研究了女贞子提取物对大强度耐力训练大鼠不同组织Ca2+ -ATPase活性的影响,探讨了女贞子提取物对大鼠运动能力的作用机制.结果表明:运动组和运动+女贞子组大鼠各组织Ca2+-ATPasee活性均显著低于安静组,运动+女贞子组大鼠不同组织Ca2+-ATPase活性显著高于运动组;运动+女贞子组大鼠力竭运动时间比运动组延长23.09%.女贞子提取物可以调节大鼠不同组织Ca2+ -ATPase活性,延长运动至疲劳的时间.%To study the mechanism of Ligustrum lucidum extract on the exercise performance of rat through examining the effects of Ligustrum lucidum extracts on Ca2+ -ATPase activity in different tissue of rats in endurance training. The results showed that the Ca2+ -ATPase activity in the rat tissue of exercise control group and exercise+ extract feeding group was significantly lower than that of the sedentary control group (/> increase Ca2+-ATPase activity, and extend the time from exercise to fatigue.

  4. Density functional investigation of metal encapsulated X-C12Si8 heterofullerene (X=Li+, Na+, K+, Be2+, Mg2+, Ca2+, Al3+, Ga3+)

    The stability and the possible application of our recently reported SiC heterofullerenes inspire the investigation of their further stabilization through ion encapsulation. The endohedral complexes X-C12Si8, where X=Li+, Na+, K+, Be2+, Mg2+, Ca2+, Al3+, and Ga3+, are probed at the MPWB1K/6-311G* and B3LYP/6-311G* levels of theory. The optimized geometries show the expanding or contracting capability of C12Si8 in order to accommodate metal ion guests. The inclusion energies indicate the stability of the complexes compared to the components. Meanwhile, the calculated binding energies show the stabilization of C12Si8 through the inclusion of Be2+, Mg2+, Al3+, and Ga3+. The host-guest interaction that is probed through NBO atomic charges supports the obtained results. This study refers to 'metal ion encapsulation' as a strategy for stabilization of SiC heterofullerenes.

  5. Effects of Ca2+, Mg2+ of Foliar Application and Combined with SA on Resistance to Botrytis cinerea in Tomato Seedlings%叶面喷施Ca2+、Mg2+、SA及其组合对番茄抗灰霉病的影响

    李琳琳; 潘晓爱; 郭秋城; 易知利


    矿质元素不仅可使植物旺盛、健壮生长,而且多数元素可作为病原物营养需要或毒害作用而影响病原物的侵染扩散和繁殖,增强植物抗病能力. 选用番茄灰霉病敏感型番茄 L402 为试材,叶片施用Ca2+、Mg2+、SA及其组合处理,对番茄五叶幼苗期抗灰霉病的效果进行调查. 结果表明:叶面增施Mg2+不显著改变番茄灰霉病的病情指数,病情指数为78. 25;Mg2+与SA不同顺序复合施用的病情性指数虽低于对照,但均显著高于SA单一处理;Mg2+不仅没有提高番茄抗灰霉病的作用,也没有提高SA诱导番茄抗灰霉病的作用;Ca2+与SA不同顺序配施对番茄幼苗抗灰霉病的影响效果不同. 明确了先施Ca2+再施用SA处理抗病效果最好. 即Ca+SA处理的病情指数比SA降低17. 93 %,比SA+Ca降低13. 34 %,比单纯施Ca2+降低45. 22 %;而SA+Ca处理的病情指数与SA处理无显著差异. 说明先施Ca2+再施SA, Ca2+具有显著增强SA诱导番茄抗灰霉病的作用. 实验结果将有助于了解和提高SA诱导抗性机制,为提高番茄产量和品种提供理论基础和现实依据.%Mineral elements as importance nutrition substance can make the plants grow strong and vigoroust, meanwhile, most elements can be used as nutritional or toxic substance of patho-gens to affect its spread, infection and reproduction, in order to enhance plant disease resist-ance. The cultivated tomato 'L402', which was sensitive to Botrytis cinerea, was employed in this experiment. In the experimet, the treatments of with Ca2+, Mg2+, SA and combinations was performed. The treatments effect on resistance on Botrytis cinerea inoculation in tomato five-leaves-stage seedlings were surveied. It was clear that the disease index was 78. 25, which was not significantly changed by foliar applying Mg2+ compared with CK. The disease index of Mg2+ application combined SA were lower than control, but significantly higher than SA single treatment. The results

  6. Effect of Turmeric, Turmerin and Curcumin on Ca2+, Na/K+ Atpases in Concanavalin A-Stimulated Human Blood Mononuclear Cells

    Suman K. Das


    Full Text Available Abstract: Ion transport enzymes may play an important role in T cell activation. This study investigates the role of turmeric and its individual components, turmerin-and curcumin-on Ca2+ and Na/K+ adenosine triphosphatases (ATPase in the course of T cell activation. Concanavalin A (Con A stimulated human blood mononuclear T cell proliferation paradigm was investigated for 3, 5 and 7 day periods with different concentrations of turmeric, curcumin and turmerin. Con A-stimulated cells treated with turmeric (250, 50, 5 μg/ml for 3 and 5 days inhibited ATPase levels when compared to base levels obtained by cells in media alone. At day 7, there was a 3-fold increase for Ca2+ATPase levels and a 2-fold increase for Na/K+ATPase. Curcumin (250, 50, 5 μg/ml showed the same pattern for ATPase activity as turmeric at 3 and 5 days with a 2-fold increase at day 7. Turmerin (2500, 1250, 250, 25 ng/ml for Na/K+ ATPase activity showed an increase at day 3, a decrease on day 5, and a 2-fold increase on day 7. Ca2+ ATPase activity in the presence of turmerin showed an increase in ATPase levels at day 3 (except at 2500ng/ml where it decreased and a decrease in day 5 (except at 25 ng/ml where it increased. Turmeric and curcumin generally inhibited Ca2+ATPase and Na/K+ATPases in early (day 3 and intermediate (day 5 stages of mitogen stimulation. However, the effect after 7 days incubation for turmeric, curcumin and turmerin showed a marked increase up to three fold.

  7. Synchronous In Situ ATPase Activity, Mechanics, and Ca2+ Sensitivity of Human and Porcine Myocardium

    Griffiths, P. J.; Isackson, H.; Pelc, Radek; Redwood, C.S.; Funari, S.S.; Watkins, H.; Ashley, C. C.


    Roč. 97, č. 9 (2009), s. 2503-2512. ISSN 0006-3495 R&D Projects: GA MŠk(CZ) LC06063 Grant ostatní: EC(XE) RII3-CT-2004-506008 Institutional research plan: CEZ:AV0Z50110509 Keywords : myocardium * actomyosin-ATPase * synchrotron-radiation Subject RIV: ED - Physiology Impact factor: 4.390, year: 2009

  8. Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

    A.C.O. Carreira


    Full Text Available The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E and aspartyl (D residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351 phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351 moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate.

  9. Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs*

    Winther, Anne-Marie L.; Liu, Huizhen; Sonntag, Yonathan; Olesen, Claus; le Maire, Marc; Soehoel, Helmer; Olsen, Carl-Erik; Christensen, S. Brøgger; Nissen, Poul; Møller, Jesper V.


    Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca2+-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed b...

  10. [Changes of sarcolemma Na+/K+ ATPase and sarcoplasmic reticulum membrane Ca2+ ATPase activity after stem cell transplantation in chronic heart failure].

    Fan, Zhongcai; Chen, Mao; Deng, Juelin; Liu, Xiaojing; Zhang, Li; Rao, Li; Yang, Qing; Huang, Dejia


    To assess the changes of sarcolemma Na+/K+ ATPase (CMNKA) and sarcoplasmic reticulum membrane Ca2+ ATPase (SERCA) activities after stem cells transplantation in heart failure. Rabbit was used as heart failure model by intravenously injecting adriamycin. Autologous bone marrow mononuclear cells (BMCs), bone marrow mesenchymal stem cells (MSCs) or skeletal myoblasts (SMs) were introduced into coronary arteies through the root of aorta when two balloons occluding just above sinus of Valsalva. After 4 weeks, left ventricular ejection fraction (LVEF)was evaluated by echocardiography, and the activities of CMNKA and SERCA were measured by colorimeter. In BMCs (n=8)and MSCs (n=8) group, LVEF were significantly improved (P SMs group (n=6) compared to sham group (n=8). The CMNKA activity in all stem cells groups was significantly increased compared to sham group (P < 0.05). Meanwhile, in comparison with sham group, the incremental tendencies of SERCA activity were seen in stem cells groups. In conclusion, stem cells transplantation could increase the activities of CMNKA and SERCA in heart failure, a possible mechanism to improve heart function. PMID:17333908

  11. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica


    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression. PMID:21531199

  12. 不同冷敏感型甘蔗茎尖Ca2+和Ca2+-ATP酶活性对低温的响应%Response of Ca2+ and Ca2+-ATPase activity in the stem tip of sugarcane to low temperature stress

    李素丽; 杨丽涛; 李志刚; 李杨瑞; 韩春旺; 梁兆宙


    为了探明不同冷敏感型甘蔗品种茎尖Ca2+和Ca2+-ATP酶活性对低温胁迫的响应机制,本研究利用植物组织化学技术结合电子显微镜观察了2个不同冷敏感型甘蔗品种茎尖在低温胁迫前后Ca2+和Ca2+-ATP酶的变化规律.供试品种为桂糖28号(抗冷型)和园林6号(冷敏感型),在0℃条件下,分别处理0、2、4和6 d后切取茎尖进行组织化学定位,获得如下结果:1)低温胁迫前后桂糖28号形态和细胞结构变化不明显,但园林6号发生质壁分离现象,线粒体空泡化,细胞崩溃,组织水溃状明显;2)低温胁迫开始,2个甘蔗品种细胞质和细胞核Ca2+沉淀颗粒均增多,但随着低温胁迫时间的延长桂糖28号细胞质和细胞核Ca2+沉淀颗粒减少,并维持在一个低稳态水平,而园林6号细胞质和细胞核一直维持在高Ca2+浓度水平;3)低温对桂糖28号Ca2+-ATP酶活性与分布影响不大,其活性一直维持在较高水平,而园林6号Ca2+-ATP酶活性随着低温胁迫时间的延长而变弱.结果说明,低温条件下细胞质和细胞核Ca2+浓度增高是导致甘蔗茎尖细胞受伤害的重要原因,维持较高Ca2+-ATP酶活性有助于避免Ca2+-中毒.%Low temperature in winter is an adverse abjotic stress to sugarcane industry,and caused significant lose. In order to investigate the response of Ca2+ and Ca2+-ATPase activity in the stem tip of different cold sensitivity sugarcane cultivars, Electron microscope (EMS) and phytohistochemistry technology were employed to detect the changes of Ca2+ level and Ca2+ -ATPase activity in the stem tips of two sugarcane varieties YL6 (cold sensitive) and GT28 (cold resistant) before and after low temperature treatment. The plants were treated at 0 ℃ in fridge and samples were taken after treatment at 0,2,4 and 6 d, respectively. The results showed that there was no significant morphological difference and cellular structures in the stem tip of GT28 before and after low

  13. Relationship between serum adiponectin level and ATPase activity of erythrocyte membrance in patients with 2-type diabetes

    Objective: To explore the possible mechanism of development nephrosis as related to changes of serum adiponectin levels and alteration of activities of Na+·K+-ATPase and Ca+2·Mg+2-ATPase of erythrocyte membrance in patients with 2-type diabetes. Methods: Serum adiponectin levels (with RIA) and erythrocyte membrance (prepared with Reilnila method) Na+·K+- ATPase and Ca+2·Mg+2-ATPase activity were determined in 45 DM2 patients without nephropathy, 31 DM2 patients with nephropathy and 30 controls. Results: Serum adiponectin levels in the diabetic patients were significantly lower than those in controls (P+·K+-ATPase and Ca+2·Mg+2-ATPase activities were also significantly lower than those in controls (P+·K+-ATPase and Ca+2·Mg+2-ATPase activities of erythrocyte membrance. (authors)

  14. [Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands].

    Menzikov, S A; Menzikova, O V


    Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate. PMID:17147268

  15. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology



    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  16. Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca2+-ATPase in cardiomyocytes of Adriamycin-treated rats

    CAI Wei; CHEN Jun-zhu; RUAN Li-ming; WANG Yi-na


    Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnI. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnI and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

  17. Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in rat cardiac pressure-overload hypertrophy


    Aim: To observe effects of angiotensin (Ang) Ⅱ receptor antagonist (AT1) irbesartan and angiotensin-converting enzyme (ACE) inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in the model of pressure-overload cardiac hypertrophy. Methods: Forty male adult Sprague Dawley rats were divided into 5 groups.One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1: sham group, rats (n=8) were gavaged with normal saline 2 ml/(kg·d)(ig); Group 2: control group, rats (n=8) were treated with normal saline 2 ml/(kg·d) (ig); Group 3: rats (n=8) were given perindopril 2 mg/(kg·d) (ig); Group 4: rats (n=8) were treated with irbesartan 20 mg/(kg·d) (ig); Group 5: rats (n=8) were given irbesartan 20 mg/(kg·d) plus perindopril 2 mg/(kg·d) (ig). Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Results: Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca2+-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca2+-ATPase. Conclusion:These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca2+-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of

  18. The investigation for the relationship among serum leptin, erythrocyte membrane Ca2+-ATPase activity and hypertensive disorder complicating pregnancy

    Chunfang Li; Wenli Gou; Xuelian Chen; Shuping Zhang


    Objective: To study the significance of Leptin and the activity of erythrocyte membrane Ca2+-ATPase (EMCA) in the development of hypertensive disorder complicating pregnancy. Methods: Radioimmunoassay was used to test the level of serum Leptin,and the activity of EMCA was determined chemically in 38 pregnant women with hypertensive disorder complicating pregnancy and 36 normotensive pregnant women. Results: The level of serum Leptin in hypertensive disorder complicating pregnancy(gestational hypertension: 13.76 ± 3.46 ng/ml; preeclampsia:15.76 ± 5.47 ng/ml; eclampsia: 18.32 ± 6.38 ng/ml)was significantly higher than that in normotensive pregnant women (11.33 ± 2.93 ng/ml), respectively. The average EMCA activity of patients with hypertensive disorder complicating pregnancy (gestational hypertension: 1.65 ± 0.24 μmol· pi/mg·h; preeclampsia: 1.37 ± 0.19 μmol·pi/mg·h; eclampsia:1.12 ± 0.14 μ mol·pi/mg·h) was significantly lower than that of normotensive pregnant women(1.83 ±0.38 μ mol·pi/mg·h),respectively. There was a negative correlation between the level of serum Leptin and the activity of RMCA in hypertensive disorder complicating pregnancy (r = -0.63). Conclusion: Inhibition of EMCA activity of erythrocyte in hypertensive disorder complicating pregnancy may increase cytoplasmic free calcium, which contributes to the development of hypertensive disorder complicating pregnancy. The negative correlation between the level of serum Leptin and the activity of EMCA, also suggested that serum Leptin and the activity of EMCA may play a role in the development of hypertensive disorder complicating pregnancy.

  19. Effect of Turmeric, Turmerin and Curcumin on Ca2+, Na/K+ Atpases in Concanavalin A-Stimulated Human Blood Mononuclear Cells

    Das, Suman K.; Michael F. Angel; Wilson, Melanie T.; Anelle Taylor; Babu Patlolla; Vijaya K. Kanji; Maheshwara-Rajeswara Rao; Cohly, Hari H.P.


    Abstract: Ion transport enzymes may play an important role in T cell activation. This study investigates the role of turmeric and its individual components, turmerin-and curcumin-on Ca2+ and Na/K+ adenosine triphosphatases (ATPase) in the course of T cell activation. Concanavalin A (Con A) stimulated human blood mononuclear T cell proliferation paradigm was investigated for 3, 5 and 7 day periods with different concentrations of turmeric, curcumin and turmerin. Con A-stimulated cells treated ...

  20. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells

    Cardozo, Alessandra K; Ortis, Fernanda; Storling, Joachim;


    , beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol......Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by...... microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding...

  1. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.


    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  2. Sarco(endoplasmic Reticulum Ca2+-ATPase-2 Gene: Structure and Transcriptional Regulation of the Human Gene

    Angel Zarain-Herzberg


    Full Text Available The sarco(endoplasmic reticulum Ca2+-ATPases (SERCAs belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5’ -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE. There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5’-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.

  3. Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

    Rice, W J; Young, H S; Martin, D W; Sachs, J R; Stokes, D.L.


    Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the cry...

  4. Stimulating effect of low doses of ionizing radiation on the activity of chicken liver and spleen plasma membrane Ca+2 ATPase during different periods of development

    Effect of pre incubative irradiation of chickens on the activity of chicken liver and spleen plasma membrane Ca+2-ATPase in 13, 15, 17 day embryos and 1, 5, 10, 20, 30, 40, 50, 60 day chickens has been studied. Low doses of radiation are discovered to stimulate liver and spleen enzyme activity. On the basis of data obtained it is suggested that in the cells of radiosensitive and radio resistive organs molecular mechanisms of stimulating effect of low doses are similar. (author). 10 refs.; 1 fig.; 2 tabs

  5. Influence of a protein hydrolysate from green algae on the activity of some ATPase systems in frog skeletal muscle.

    Ivanov, R; Georgieva, B; Naumova, P; Mileva, K; Radicheva, N


    The present study investigated the effect of a protein hydrolysate from green algae cultured in the Bulgarian region of Rupy, on the enzyme activity of frog skeletal muscle. The activity of pure Mg(2+)-ATPase, Mg2+,Ca(2+)-ATPase, NaHCO3-stimulated Mg(2+)-ATPase and the latter in the presence of the inhibitors NaSCN and NaN3 in mitochondrial (B-3) and membrane (B-12) fractions were determined before and after treatment with the protein hydrolysate from green algae (30 and 300 micrograms/ml). The differences between ATPase activity of mitochondrial and membrane fractions were described and it was established that in the B-3 fraction, the activity of the NaHCO3-stimulated Mg(2+)-ATPase and Ca(2+)-dependent Mg(2+)-ATPase were accelerated by increasing concentrations of the algae protein hydrolysate. Irrespective of the different (equal or inverse) dose-dependent effects, the protein hydrolysate stimulated Mg(2+)-ATPase and that inhibited by NaSCN an NaN3 bicarbonate-stimulated Mg(2+)-ATPase activity. In most of the probes, the protein hydrolysate produced some increase in enzyme activity of NaHCO3-stimulated Mg(2+)-ATPase and Ca(2+)-dependent Mg(2+)-ATPase in B-12 fractions. The observed properties of the algae protein hydrolysate suggest that it is capable of stimulating enzyme processes in addition to having some antitoxic effect in skeletal muscle. PMID:10420389

  6. Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.

    Chao, A C; Kouyama, K; Heist, E K; Dong, Y. J.; Gardner, P


    Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-de...

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

    Roewer Norbert


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

  8. X-ray effects on the activity of a Mg2+-dependent, Na+- and K+-activable microsomal membrane ATP-ase system

    The bahviour of a Mg2+-dependent, Na+- and K+-activable ATP-ase sytem on irradiation was investigated using a microsome fraction of guinea pig myocardial cells prepared by fractionated centrifugation. The Na+- and K+-activable component, transport-ATPase, was particularly radiation-sensitive. Three stages of development were observed for a 1,500 R radiation damage until 24 h p.r.. In the first stage, until 30 minutes p.r., the activity of transport-ATP-ase was inhibited. This was followed by repair processes which had reached a peak value clearly higher than the control values at 4 hours p.r.. In the third stage, the activity was reduced again; 15 and 24 hours after termination of exposure, values again were nearly the same as after 30 minutes where a maximum was observed for this radiation dose. Radiation-induced electrolyte displacements, active transport, and radiation-induced inhibition of transport-ATP-ase were correlated and discussed; the assumption was that changes in, the electrolyte conditions in the membranes on irradiation are at least partly due to the described inhibition of transport-ATP-ase. (orig./AJ)

  9. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase

    Highlights: ► Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. ► ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. ► Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. ► Subcellular localization of SPCA2 may depend on tissue type. ► In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

  10. The calcium pump plasma membrane Ca(2+)-ATPase 2 (PMCA2) regulates breast cancer cell proliferation and sensitivity to doxorubicin.

    Peters, Amelia A; Milevskiy, Michael J G; Lee, Wei C; Curry, Merril C; Smart, Chanel E; Saunus, Jodi M; Reid, Lynne; da Silva, Leonard; Marcial, Daneth L; Dray, Eloise; Brown, Melissa A; Lakhani, Sunil R; Roberts-Thomson, Sarah J; Monteith, Gregory R


    Regulation of Ca(2+) transport is vital in physiological processes, including lactation, proliferation and apoptosis. The plasmalemmal Ca(2+) pump isoform 2 (PMCA2) a calcium ion efflux pump, was the first protein identified to be crucial in the transport of Ca(2+) ions into milk during lactation in mice. In these studies we show that PMCA2 is also expressed in human epithelia undergoing lactational remodeling and also report strong PMCA2 staining on apical membranes of luminal epithelia in approximately 9% of human breast cancers we assessed. Membrane protein expression was not significantly associated with grade or hormone receptor status. However, PMCA2 mRNA levels were enriched in Basal breast cancers where it was positively correlated with survival. Silencing of PMCA2 reduced MDA-MB-231 breast cancer cell proliferation, whereas silencing of the related isoforms PMCA1 and PMCA4 had no effect. PMCA2 silencing also sensitized MDA-MB-231 cells to the cytotoxic agent doxorubicin. Targeting PMCA2 alone or in combination with cytotoxic therapy may be worthy of investigation as a therapeutic strategy in breast cancer. PMCA2 mRNA levels are also a potential tool in identifying poor responders to therapy in women with Basal breast cancer. PMID:27148852

  11. Effects of Sodium Sulfate and Sodium Chloride on Ca2+ , Mg2+ Removal from Glauber Type Brine by Sodium Hydroxide and Sodium Carbonate%芒硝型卤水中盐硝组分对碱法脱除钙镁的影响

    董泽亮; 张琦; 王俐聪; 蔡荣华; 马来波; 黄西平


    Ca2+ 、Mg2+ removal from Glauber type brine by sodium hydroxide and sodium carbonate has been studied.The effects of sodium sulfate and sodium chloride on removal efficiency of Ca2+,Mg2+ at room temperature were investigated in detail when addition amount of sodium hydroxide and sodium carbonate was theoretical value,reaction time was 30 min,aging time was 60 min.The experimental results show that the presence of sodium sulfate has large effect on removal efficiency of Ca2+.The removal rate of Ca2+ is more than 90% when the concentration of sodium sulfate is below 30 g/L.The presence of sodium sulfate has good but little effect on removal efficiency of Mg2+.The presence of sodium chloride has small effect on removal efficiency of Ca2+ and Mg2+.When the concentration of sodium chloride increases,the removal rate of Ca2+ increases significantly,but the removal rate of Mg2+ declines slightly.%采用“烧碱-纯碱”法,对芒硝型卤水中Ca2+和Mg2+的脱除进行了研究,在常温、两碱用量为理论用量、反应时间为30 min和陈化时间为60 min的条件下详细考察了卤水中氯化钠和硫酸钠含量的变化对Ca2+和Mg2脱除效果的影响.结果表明,硫酸钠组分对Ca2+的脱除效果影响较大,当硫酸钠含量在30 g/L以下时,Ca2+脱除率在90%以上,硫酸钠浓度增加有利于Mg2+的脱除,但影响不大.氯化钠组分对Ca2Mg2+脱除效果的影响相对较小,氯化钠含量增加,Ca2+脱除率明显增加,而Mg2+脱除率略有下降.

  12. Effect of major cation water composition on the ion exchange of Np(V) on montmorillonite: NpO2+–Na+–K+–Ca2+–Mg2+ selectivity coefficients

    Highlights: • Determined Np(V)-montmorillonite ion exchange constant, applicable in a wide range of conditions. • Developed a model for Np(V) ion exchange which can be readily applied in thermodynamic databases. • Identified solution conditions at which Np(V) ion exchange will play a significant role. - Abstract: Np(V) sorption was examined in pH 4.5 colloidal suspensions of nominally homoionic montmorillonite (Na-, K-, Ca- and Mg-montmorillonite). Ionic exchange on permanent charge sites was studied as a function of ionic strength (0.1, 0.01 and 0.001 M) and background electrolyte (NaCl, KCl, CaCl2 and MgCl2). An ion exchange model was developed using the FIT4FD program, which considered all experimental data simultaneously: Np sorption data, major cation composition of the electrolyte and associated uncertainties. The model was developed to be consistent with the ion exchange selectivity coefficients between the major cations reported in the literature and led to the following recommended selectivity coefficients for Np(V) ion exchange according to the Vanselow convention: log(NpO2+Na+KV)=-0.20,log(NpO2+K+KV)=-0.46,log(NpO2+Ca2+KV)=-0.57,log (NpO2+Mg2+KV)=-0.57. Both the experimental data and the estimated selectivity coefficients in this study are consistent with the limited Np(V) ion exchange and sorption data reported in the literature. The results indicate that, as expected, low ionic strengths favor Np(V) sorption when ion exchange is the main sorption mechanism (i.e. acidic to neutral pHs) and that the divalent cations Ca2+ and Mg2+ may be important in limiting Np(V) ionic exchange on montmorillonite

  13. Geographic structuring of the Plasmodium falciparum sarco(endoplasmic reticulum Ca2+ ATPase (PfSERCA gene diversity.

    Ronan Jambou

    Full Text Available Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp, of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates. This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

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

    Wei XIN


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

  15. Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques

    The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism

  16. Relationship between changes of plasma endothelin (ET) level, ATPase activity of erythrocyte membrane and development of nephropathy in patients with pregnancy induced hypertension

    Objective: To investigate the possible role played by alteration of plasma ET levels and activities of Na+- K+-APT ase and Ca2+-Mg2+-ATPase of erythrocyte membrane in patients with nephropathy pregnancy induced hypertension. Methods: The concentrations of plasma ET was detected with RIA and erythrocyte membrane ATPase activities were determined with Reilni method in 32 pregnant women with PIH complicated with nephropathy and 70 women with PIH but no nephropathy and 35 normal pregnant women as controls. Results: The plasma ET levels in patients with PHI (both with and without nephropathy) were significantly higher than those in normal preganat women (P+-K+-ATPase and Ca2+-Mg2+-ATPase levels were significantly de- creased (P+-K+-ATPase and Ca2+-Mg2+-ATPase activity of erythrocyte membrane. (authors)

  17. Total soil electrical conductivity and critical soil K+ to Ca2+ and Mg2+ ratio for potato crops Condutividade elétrica e níveis críticos da relação entre K+ e Ca+ + Mg+ no solo para cultura da batata

    Roberto Anjos Reis Jr.; Paulo Cezar Rezende Fontes; Júlio Cesar Lima Neves; Nerilson Terra Santos


    Soil K+ to Ca2+ and Mg2+ ratio as well as the total salinity were evaluated in response to potassium fertilizer application onto potato. Potassium was applied at six different rates (0, 60, 120, 240, 480 and 960 kg ha-1 of K2O), as K2SO4, and was placed during planting time in the furrow. Soil from the 0-200 mm layer was collected in the furrow, 20 and 48 days after plant emergence (DAE) to evaluate soil pH, K+, Ca2+ and Mg2+ contents and the total electrical conductivity (EC). A factorial de...

  18. Sub-chronic effect of neem based pesticide (Vepacide) on acetylcholinesterase and ATPases in rat.

    Rahman, M F; Siddiqui, M K; Jamil, K


    Acetylcholinesterases (AChE), Na(+)-K+, Mg2+ and Ca(2+)-ATPases were monitored in rat brain when treated orally with 80, 160 and 320 mg/kg of Vepacide, an active ingredient from neem seed oil, daily for 90 days. Brain AChE, Na(+)-K+ and Ca(2+)-ATPases were inhibited whereas Mg(2+)-ATPase levels were enhanced in both the sexes after 45 and 90 days of treatment. The relative sensitivities of these ATPases to Vepacide indicated that Ca(2+)-ATPase being more sensitive than Na(+)-K(+)-ATPase in both the sexes. The magnitude of Ca(2+)-ATPase inhibited by this compound was higher than that of brain AChE. It appears to be sexual dimorphism in the alterations of brain AChE, Na(+)-K+ and Mg(2+)-ATPases by Vepacide with females being significant when compared with males. After 28 days of post treatment the alterations observed were approached to those of controls both in male and female rats showing reversal of the toxicity. These results indicated that the ATPases were potently inhibited by Vepacide and seemed to be its precise target among the enzyme studied. This can be used as biochemical marker of exposure to this neem derived product. PMID:10466107

  19. Chelating agents related to ethylenediamine bis(2-hydroxyphenyl)acetic acid (EDDHA): synthesis, characterization, and equilibrium studies of the free ligands and their Mg2+, Ca2+, Cu2+, and Fe3+ chelates.

    Yunta, Felipe; García-Marco, Sonia; Lucena, Juan J; Gómez-Gallego, Mar; Alcázar, Roberto; Sierra, Miguel A


    Iron chelates such as ethylenediamine-N,N'-bis(2-hydroxyphenyl)acetic acid (EDDHA) and their analogues are the most efficient soil fertilizers to treat iron chlorosis in plants growing in calcareous soils. EDDHA, EDDH4MA (ethylenediamine-N,N'-bis(2-hydroxy-4-methylphenyl)acetic acid), and EDDCHA (ethylenediamine-N,N'-bis(2-hydroxy-5-carboxyphenyl)acetic acid) are allowed by the European directive, but also EDDHSA (ethylenediamine-N,N'-bis(2-hydroxy-5-sulfonylphenyl)acetic acid) and EDDH5MA (ethylenediamine-N,N'-bis(2-hydroxy-5-methylphenyl)acetic acid) are present in several commercial iron chelates. In this study, these chelating agents as well as p,p-EDDHA (ethylenediamine-N,N'-bis(4-hydroxyphenyl)acetic acid) and EDDMtxA (ethylenediamine-N,N'-bis(2-metoxyphenyl)acetic acid) have been obtained following a new synthetic pathway. Their chemical behavior has been studied to predict the effect of the substituents in the benzene ring on their efficacy as iron fertilizers for soils above pH 7. The purity of the chelating agents has been determined using a novel methodology through spectrophotometric titration at 480 nm with Fe(3+) as titrant to evaluate the inorganic impurities. The protonation constants were determined by both spectrophotometric and potentiometric methods, and Ca(2+) and Mg(2+) stability constants were determined from potentiometric titrations. To establish the Fe(3+) and Cu(2+) stability constants, a new spectrophotometric method has been developed, and the results were compared with those reported in the literature for EDDHA and EDDHMA and their meso- and rac-isomers. pM values have been also determined to provide a comparable basis to establish the relative chelating ability of these ligands. The purity obtained for the ligands is higher than 87% in all cases and is comparable with that obtained by (1)H NMR. No significant differences have been found among ligands when their protonation and stability constants were compared. As expected, no Fe(3

  20. 女贞子提取物对大强度耐力训练大鼠不同组织Mg2+-ATPase活性的影响%Effect of Fructus Ligustri Lucidi Extracts on Mg2+-ATPase Activity in Different Tissues of Rats of High-Intensity Endurance Training and Exercise Capacity

    马云慧; 熊正英


    To analyze the change of Mg2+-ATPase activity in different tissue of rats like heart,liver,brain,kidney and quadriceps after the rats were fed with Fructus Ligustri Lucidi extracts (FLLE) and trained with high-intensity endurance training.To study the effect of FLLE as the exercise nutrition tonifying formula on the exercise capacity in rats.SD rats were randomly divided Sedentary control group,exercise control group and exercise + FLLE group,n =8.Exercise control group received high-intensity treadmill training for 6 weeks,exercise+FLLE group was fed with 2mL 400 mg/kg FLLE extract besides high-intensity treadmill training daily.Sedentary control group and exercise control group was given with 2 mL 0.5 % Tween-80 solution.After 6 weeks,sedentary control group was in rest state,and after exercise control and exercise+FLLE group were given an exhaustive exercise,determination of the different tissue Mg2+-ATPase activity of each group.The results show the exercise control group and exercise+ FLLE group of the different tissue Mg2+-ATPase activity was significantly lower than the sedentary control group,exercise+FLLE group of the different tissue Mg2+-ATPase activity was higher than the exercise control group (P<0.01 or P<0.05); with the sedentary control group,exercise control rat heart,liver,brain,kidney and quadriceps Mg2+-ATPase activity decreased by 21.01%,10.06 %,11.15 %,19.89 % and 12.36 %; with the exercise control group,exercise+FLLE group rat heart,liver,brain,kidney and quadriceps Mg2+-ATPase activity were increased by 21.62%,9.21%,8.24%,21.94% and 7.05%; compared with that of rats in the control group,the time of exhaustive exercise of rats in the exercise+FLLE group was prolonged by 23.09 %.That indicates,complementing FLLE can increase the concentration of antioxidants in the body,preventing oxidative damage to cell membranes,maintaining the steady-state concentrations of intracellular ion,and ensuring the mitochondria

  1. Total soil electrical conductivity and critical soil K+ to Ca2+ and Mg2+ ratio for potato crops Condutividade elétrica e níveis críticos da relação entre K+ e Ca+ + Mg+ no solo para cultura da batata

    Roberto Anjos Reis Jr.


    Full Text Available Soil K+ to Ca2+ and Mg2+ ratio as well as the total salinity were evaluated in response to potassium fertilizer application onto potato. Potassium was applied at six different rates (0, 60, 120, 240, 480 and 960 kg ha-1 of K2O, as K2SO4, and was placed during planting time in the furrow. Soil from the 0-200 mm layer was collected in the furrow, 20 and 48 days after plant emergence (DAE to evaluate soil pH, K+, Ca2+ and Mg2+ contents and the total electrical conductivity (EC. A factorial design (6x2, with six K rates and two sampling times was set up in a randomized block design with four replications. The application of K fertilizer increased exchangeable K, did not affect pH and exchangeable Ca and Mg contents, but caused a linear increase of the soil K+/(Ca2++Mg2+1/2 ratio as well as EC. At 20 DAE, the critical soil K+/(Ca2++Mg2+1/2ratio and the EC associated with maximum tuber yield (30.5 Mg.ha-1, with 353.4 kg ha-1 of K2O were 1.79 and 1.6 dS m-1, respectively. The highest soil K+/(Ca2++Mg2+1/2 ratio and EC were obtained with the highest application of K fertilizer, which led to a reduction in the potato tuber yield.Com o objetivo de avaliar a relação entre K e Ca + Mg e a salinidade no solo em resposta à adubação potássica no cultivo da batateira (cultivar Baraka, foi instalado experimento fatorial a nível de campo com seis doses de potássio (0, 60, 120, 240, 480 e 960 kg ha-1 de K2O e duas épocas de amostragem, 20 e 48 dias após a emergência das plantas, (DAE delineado em blocos casualizados com quatro repetições. O potássio foi aplicado como K2SO4 no sulco de plantio. O solo foi amostrado (0-200 mm de profundidade para avaliar o pH, a condutividade elétrica e os teores de K, Ca e Mg. A adubação potássica aumentou o K trocável, não afetou o pH e os teores de Ca e Mg trocáveis no solo, e elevou linearmente a condutividade elétrica e a relação K+/(Ca2++ Mg2+1/2. Aos 20 DAE, a máxima produção de tubérculos foi

  2. Hailey-Hailey disease and tight junctions: Claudins 1 and 4 are regulated by ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 in cultured keratinocytes

    Raiko, Laura; Siljamäki, Elina; Mahoney, Mỹ G.; Putaala, Heli; Suominen, Erkki; Peltonen, Juha; Peltonen, Sirkku


    Mutations in the ATP2C1 gene encoding Ca2+/Mn2+ ATPase SPCA1 cause Hailey-Hailey disease (HHD, OMIM 16960). HHD is characterized by epidermal acantholysis. We attempted to model HHD using normal keratinocytes in which the SPCA1 mRNA was down-regulated with the small inhibitory RNA (siRNA) method. SiRNA inhibition significantly down-regulated the SPCA1 mRNA, as demonstrated by qPCR, and decreased the SPCA1 protein beyond detectable level, as shown by western analysis. The expression of selecte...

  3. Antioxidation and ATPase activity in the gill of mud crab Scylla serrata under cold stress

    KONG Xianghui; WANG Guizhong; LI Shaojing


    Mud crab (Scylla serrata) is an important commercial crustacean in China. An experiment was designed to study the effect of cold stress on S. serrata. After a one-week adaptation at 28 ℃, the temperature is suddenly reduced to 4 ℃. The crabs were sampled every 2 h for 10 h and dissected immediately to measure the enzyme activity. The crabs at room temperature (28 ℃) were used as the control group. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the content of malondialdehyde (MDA) and the activity of 4 ATPases (Na+, K+-ATPase;Mg2+-ATPase; Ca2+-ATPase; Ca2+, Mg2+-ATPase) were measured biochemically. In contrast to the control group, the SOD activity increased significantly from 2 to 6 h after the cold stress, and then decreased. The CAT and GPX activities increased in 2 h, and then decreased gradually. The content of MDA increased gradually in 4 h. The activity ofNa+, K+-ATPase decreased in 2 h, increased up to the top value at Hour 6,then decreased again. The activities of Mg2+-ATPase, Ca2+-ATPase and Ca2+, Mg2+-ATPase increased significantly in 6 h, insignificantly in any other hours. Under cold stress, the activity of antioxidative enzymes in S. serrata was reduced at first then stabilized, ROS-scavenging weakened, and MDA accumulated gradually in the gill after 6 h. The activity of the 4 ATPases in the crab decreased after 6 h,suggesting that the ability to regulate ion concentration has been paralyzed. Therefore, the maximum period to sustain healthy meat in the crab under cold stress is 6 hours.

  4. Effects of Four Interior-warming Drugs on the Tension of Ileum Smooth Muscle and Ca2+-ATPase in Rabbits%4种温里药对兔回肠平滑肌张力及Ca2+-ATP 酶的影响

    黄庆芳; 陈艳芬; 杨全; 杨超燕; 唐春萍; 明露; 黎洁玲; 陶曙红


    Objective To investigate the effects of four interior-warming drugs( galangal,cinnamon,evodia rutaecarpa,and dried ginger)on the tension of ileum smooth muscle and Ca2+-ATPase on the cell membrane in rabbits. Methods The effects of galangal,cinnamon,evodia rutaecarpa,and dried ginger were examined on normal ileum smooth muscle,in vitro intestinal muscle contraction caused by acetylcholine(ACh),barium chloride(BaCl2 )and histamine(His), and ACh-induced calcium release by using BL-420E+ biological signal collection and processing system.The average tension was measured within 1 min before delivery and within 3 minutes after the treatment,and the inhibition rate was calculated according to the average tension value.The effects of sera containing galangal,cinnamon,evodia rutaecarpa,and dried ginger on Ca2+-ATPase activity on the cell membrane of the intestinal smooth muscle were examined by phosphorus method. Results Galangal,cinnamon,evodia rutaecarpa,and dried ginger at high concentrations could restrain in vitro intestinal contraction in normal circumstances(P<0.05 or P<0.01).Significant inhibitory effects on intestinal contraction caused by ACh,His and BaCl2 were found in low,medium and high concentration groups(P<0.01).There was a dose-effectiveness relationship between the inhibition rate and final drug concentrations.The ACh-induced intracellular and extracellular calcium dependent contraction were significantly inhibited by the four interior-warming drugs( P < 0. 05 or P < 0. 01). The Ca2+-ATPase activities were( 0. 384 ± 0.070),(0.302±0.016),(0.307±0.016),(0.296±0.016),(0.313±0.003)U·mg-1 ,respectively,in intestinal smooth muscle in normal control group and high concentration groups of galangal,cinnamon,evodia rutaecarpa,and dried ginger(P<0.01). Conclusion Interior-warming drugs may relax intestinal smooth muscle by reducing the intracellular calcium release and the extracellular calcium inflow via receptor-controlled calcium channels,and inhibiting the

  5. Function and Evolution of a MicroRNA That Regulates a Ca2+-ATPase and Triggers the Formation of Phased Small Interfering RNAs in Tomato Reproductive Growth[W][OA

    Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao


    MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca2+-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca2+-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species. PMID:21917547

  6. 脱硫废弃物对碱胁迫下水稻叶片钙分布、Ca2+-ATPase活性及抗氧化特征的影响%Effects of desulfurization waste on calcium distribution, Ca2+-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress

    毛桂莲; 许兴; 曾瑾; 岳自慧; 杨淑娟


    为了探讨脱硫废弃物提高水稻抗盐碱的作用机制,采用盆栽法,研究脱硫废弃物对碱胁迫下水稻幼苗叶片总钙含量、Ca2+分布、细胞膜Ca2+-ATPase活性及活性氧含量等的变化.结果表明:对照处理的细胞中钙颗粒零星分布于细胞壁和叶绿体中,添加脱硫废弃物和CaSO4处理的细胞质膜、细胞间隙、细胞壁和液泡中有大量的钙颗粒分布;随着脱硫废弃物和CaSO4添加量的增加,叶片总钙含量增加,质膜和液泡膜Ca2+-ATPase活性呈上升趋势,质膜透性、MDA含量和活性氧O2-产生速率呈下降趋势,SOD、POD等保护酶活性升高.添加脱硫废弃物在一定程度上能够减缓碱胁迫对水稻造成的细胞伤害,起主要作用的物质可能是其主要成分CaSO4.%To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca2+-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaS04 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca2+ -ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2' production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaS04.

  7. Relationship between serum leptin levels, ATPase activity of erythrocyte membrance and development of diabetic nephropathy in patients with DM2

    Objective: To study the possible mechanism of development of nephrosis affected by changes of serum leptin levels and alteration of activities of Na+K+-ATPase and Ca2+Mg2+-ATPase of erythrocyte membrane in patients with type 2 diabetes(DM2). Methods: Serum leptin levels (with RIA) and erythrocyte membrane Na+K+-ATPase and Ca2+Mg2+-ATPase activitities (with Reinila method) were determined in 40 DM2 patients without nephropathy, 32 DM2 patients with nephropathy and 35 controls. Results Serum leptin levels were significantly higher in the diabetics as a whole than those in controls (P+K+-ATPase and Ca2+Mg2+-ATPase activities were significantly lower (P<0.01). Among the diabetic patients, the serum leptin levels in patients without nephrosis (P<0.05), but the RBC membrance ATPase activities were significantly lower(P<0.05). Conclusion: Development of type 2 diabetes nephrosis might be correlated with the high serum leptin level and decreased ATPase activities of erythrocite membrane. (authors)

  8. The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide.

    Petrunyaka, V V; Panyushkina, E A; Severina, E P; Orlov, S N


    The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes. PMID:2175654

  9. Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca2+-ATPase PfATP6

    Daniel Silqueira Martins Guimarães


    Full Text Available Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

  10. Materiales de Al2O3 - MgAl2O4 - CaAl12O19 - Ca2Mg2Al28O46 obtenidos mediante un proceso de sinterización reactiva entre Al2O3 y CaMg(CO32

    Pena, P.


    Full Text Available Taking into account the system Al2O3-MgO-CaO, refractory materials containing Al2O3-MgAl2O4-CaAl12O19-Ca2Mg2Al28O46 has been obtained by reaction-sintering of appropriate mixtures of Al2O3 and CaMg(CO32. The study of the mechanism of reaction was carried out by differential thermal analysis (DTA and thermogravimetry (TG, followed by dilatometric and xray diffraction (XRD studies. A static study of the reaction was performed at different temperatures. After each thermal treatment a XRD analysis of the phases present was made, as well as a microestructural study by scanning electron microscopy (SEM coupled with energy-dispersive spectroscopy (EDS. From the results obtained, and taking into account the free energy of formation of the different compounds, the mechanism of reaction was established.Utilizado la información suministrada por el diagrama de equilibrio de fases Al2O3-MgO-CaO se ha diseñado y obtenido un material de Al2O3-MgAl2O4-CaAl12O19-Ca2Mg2Al28O46 mediante sinterización reactiva de una mezcla de Al2O3 y CaMg(CO32. Las reacciones que tienen lugar en la mezcla durante el proceso se han estudiado usando técnicas de análisis térmico diferencial, termogravimetrico y dilatometría. Muestras reaccionadas a temperaturas seleccionadas se han estudiado por difracción de rayos X y microscopía electrónica de barrido con microanálisis mediante dispersión de energías. Los resultados obtenidos revelan que la reacción entre las materias primas tiene lugar en varias etapas con formación de fases transitorias, dando lugar a una microestructura final con granos aciculares de CaAl12O19 y partículas de la fase ternaria Ca2Mg2Al28O46 formadas en aquellos puntos en los que entran en contacto las fases de MgAl2O4 y CaAl12O19.

  11. Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles


    A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fuel...

  12. Crystal Structure of the Vanadate-Inhibited Ca2+-ATPase

    Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand Jean-Paul; Olesen, Claus; Andersen, Jens Peter; Møller, Jesper Vuust; Nissen, Poul


    catalytic site as a planar VO3− in complex with water and Mg2+ in a dephosphorylation transition-state-like conformation. Validating bound VO3− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization......Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound at the...

  13. Materiales de Al2O3 - MgAl2O4 - CaAl12O19 - Ca2Mg2Al28O46 obtenidos mediante un proceso de sinterización reactiva entre Al2O3 y CaMg(CO3)2

    Aza Moya, Antonio H. de; Peña, P.; Moset, M.


    [ES] Utilizado la información suministrada por el diagrama de equilibrio de fases Al2O3-MgO-CaO se ha diseñado y obtenido un material de Al2O3-MgAl2O4-CaAl12O19-Ca2Mg2Al28O46 mediante sinterización reactiva de una mezcla de Al2O3 y CaMg(CO3)2. Las reacciones que tienen lugar en la mezcla durante el proceso se han estudiado usando técnicas de análisis térmico diferencial, termogravimetrico y dilatometría. Muestras reaccionadas a temperaturas seleccionadas se han estudiado por difracci...

  14. Secretory pathway Ca(2+)-ATPase isoform 1 knockdown promotes Golgi apparatus stress injury in a mouse model of focal cerebral ischemia-reperfusion: In vivo and in vitro study.

    Fan, Yongmei; Zhang, Changjie; Peng, Wenna; Li, Ting; Yin, Jing; Kong, Ying; Lan, Chunna; Li, Xiaofang; Wang, Rumi; Hu, Zhiping


    The present study was designed to investigate the potential role of secretory pathway Ca(2+)-ATPase isoform 1(SPCA1) in experimental focal cerebral ischemia-reperfusion injury. Cerebral ischemia-reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 2h s in Sprague-Dawley rats, and then the expression levels of SPAC1 mRNA and protein were determined. Results showed that SPCA1 level was transiently increased 1 day after reperfusion in peri-infarction area, while markedly increased in infarction core on 3day and 7 day after reperfusion. Then a SPCA1 lentivirus was used to achieve knockdown of SPCA1 gene: Ca(2+) transporting type 2C, member 1 (ATP2C1) gene. It has been observed that SPCA1 knockdown by lentivirus markedly increased cerebral infarction volume in vivo. Meanwhile, SPCA1 knockdown also facilitated per-oxidative production, including nitric oxide (NO) and 3-nitrotyrosine (3-NT) and decreased the expression of total superoxide dismutase (SOD) and manganese superoxide dismutase (MnSOD). Moreover, in vitro study showed that SPCA1 knockdown increased hydrogen peroxide (H2O2)-induced lactate dehydrogenase (LDH) leakage dose-dependently, and elevated caspase3 level in neuro-2a (N2a) cells. In addition, SPCA1 knockdown increased H2O2-induced production of nitric oxide and 3-NT dose-dependently, and reversed the increased activity of total SOD and MnSOD in neuro-2a cells. In conclusion, the present study indicated that SPCA1 could suppress over active Golgi apparatus (GA) stress thus attenuate cerebral ischemia-reperfusion injury. PMID:27038757

  15. A many-body model to study proteins. I. Applications to MLnm+ complexes, Mm+=Li+, Na+, K+, Mg2+, Ca2+, and Zn2+, L=H2O, CH3OH, HCONH2, n=1-6, and to small hydrogen bonded systems

    Masella, Michel; Cuniasse, Philippe


    A new model to study proteinic systems including a many-body polarization and a hydrogen bond energy contribution is presented. This model represents an extension of an earlier water many-body model [M. Masella and J.-P. Flament, J. Chem. Phys. 107 9105 (1997)]. As in this earlier model, the new model is developed to reproduce quantum computations on small molecular aggregates, and, in this first paper, we focus our efforts in developing an accurate potential to describe interactions among all nonbonded atoms occurring in proteins, and among those atoms and six cations of biological interest: Li+, Na+, K+, Mg2+, Ca2+, and Zn2+. Intramolecular degrees of freedom are described as in classical two-body force fields. In the present paper, the new model is applied to investigate the properties of small ion-neutral [M,Ln]m+ complexes and of small hydrogen-bonded systems. The results showed that this model is able to reproduce most of the theoretical quantum predictions and experimental data published until now regarding those systems.

  16. Motion of the Ca2+-pump captured.

    Yokokawa, Masatoshi; Takeyasu, Kunio


    Studies of ion pumps, such as ATP synthetase and Ca(2+)-ATPase, have a long history. The crystal structures of several kinds of ion pump have been resolved, and provide static pictures of mechanisms of ion transport. In this study, using fast-scanning atomic force microscopy, we have visualized conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in real time at the single-molecule level. The analyses of individual SERCA molecules in the presence of both ATP and free Ca(2+) revealed up-down structural changes corresponding to the Albers-Post scheme. This fluctuation was strongly affected by the ATP and Ca(2+) concentrations, and was prevented by an inhibitor, thapsigargin. Interestingly, at a physiological ATP concentrations, the up-down motion disappeared completely. These results indicate that SERCA does not transit through the shortest structure, and has a catalytic pathway different from the ordinary Albers-Post scheme under physiological conditions. PMID:21707923

  17. Oxidative damage increases intracellular free calcium [Ca2+]i concentration in human erythrocytes incubated with lead.

    Quintanar-Escorza, M A; González-Martínez, M T; del Pilar, Intriago-Ortega Ma; Calderón-Salinas, J V


    One important effect of lead toxicity in erythrocytes consists of increasing [Ca(2+)](i) which in turn may cause alterations in cell shape and volume and it is associated with cellular rigidity, hemolysis, senescence and apoptosis. In this work, we proposed the use of erythrocytes incubated with Pb(2+) to assess association of the mechanisms of lead erythrocyte oxidative damage and calcium homeostasis. Lead incubation produced an increase in [Ca(2+)](i) dose- and time-dependent, which mainly involved Ca(2+) entry mechanism. Additionally, in this in vitro model alterations similar to erythrocytes of lead-exposed workers were produced: Increase in Ca(2+) influx, decrease in (Ca(2+)-Mg(2+))-ATPase activity and GSH/GSGG ratio; increase in lipoperoxidation, protein carbonylation and osmotic fragility accompanied of dramatic morphological changes. Co-incubation with trolox, a soluble vitamin-E analog is able to prevent these alterations indicating that lead damage mechanism is strongly associated with oxidative damage with an intermediate toxic effect via [Ca(2+)](i) increase. Furthermore, erythrocytes oxidation induced with a free radical generator (APPH) showed effects in [Ca(2+)](i) and oxidative damage similar to those found in erythrocytes incubated with lead. Co-incubation with trolox prevents the oxidative effects induced by AAPH in erythrocytes. These results suggest that increase of [Ca(2+)](i) depends on the oxidative status of the erythrocytes incubated with lead. We consider that this model contributes in the understanding of the relation between oxidative damage induced by lead exposure and Ca(2+) homeostasis, the consequences related to these phenomena and the molecular basis of lead toxicity in no excitable cells. PMID:20460147

  18. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca2+, Na+, K+ and H+), have been reported. They include reticulum and plasma-membrane Ca2+-ATPases, Na+/K+-ATPase and H+/K+-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg2+ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na+/K+-ATPase α1-isoform, H+/K+-ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H+/K+-ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  19. Effects of exogenous creatine phosphate on glutamic acid and Ca2+-ATPase activity in brain of mice after exhaustive exercise%外源性磷酸肌酸对游泳力竭小鼠大脑中谷氨酸和钙-ATP酶活力的影响

    马集; 卢畅; 姜茜; 殷林波; 刘彦娜; 刘克敏


    Objective:To observe the effects of exogenous creatine phosphate on glutamic acid level and Ca2+-ATPase activity in brain of mice after exhaustive exercise and to further reveal the mechanism of exogenous creatine phosphate in allaying tiredness.Methods:All 36 mice,6-week-age,were divided into 4 groups:exhaustive swimming control group (group A); exhaustive swimming with medication group (group B); 8-min swimming control group (group C);and 8-min swimming with medication group (group D).The method of mice weight-loading swimming was used to sets up the model of exhaustive exercise,and each mouse loaded weight with 6% of the mass of itself.Thirty min before the experiment,mice in groups B and D were given the intraperitoneal injection with creatine phosphate sodium by the standard of 1000 mg/kg,and the mice in groups A and C were given the same proportionate normal saline as placebo.The exhaustive swimming time was recorded,and glutamic level and Ca2+-ATPase activity were measured by using biochemical kits.Results:After testing,the exhaustion time in group B was longer than that in group A (P<0.05).The Glu contents in groups B and D were significantly lower than in groups A and C (P<0.05).Ca2+-ATPase activity in groups B and group D was significantly higher than that in groups A and C (P<0.05).Conclusion:The mechanism of exogenous creatine phosphate in allaying tiredness may be closely related with increased Ca2+-ATPase activity and reduced glutamic level.%目的:研究外源性磷酸肌酸(PCr)对游泳力竭小鼠大脑中谷氨酸(Glu)和钙-ATP酶(Ca2+-ATPase)活力的影响,以进一步揭示PCr的抗疲劳机制.方法:将44只6周龄小鼠分为力竭对照组12只(A组)、力竭给药组12只(B组)、游泳8min对照组10只(C组)、游泳8min给药组10只(D组),采取小鼠负重游泳的力竭运动模型,每只小鼠负重量为自身体质量的6%.于游泳前30min,B、D组小鼠经腹腔注射磷酸肌酸钠溶液1000mg/kg;A、C组小鼠注

  20. Hg2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca2+-induced Ca2+ release.

    Burlando, Bruno; Bonomo, Marco; Fabbri, Elena; Dondero, Francesco; Viarengo, Aldo


    Mercury is a non-essential heavy metal affecting intracellular Ca2+ dynamics. We studied the effects of Hg2+ on [Ca2+]i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg2+ induced dose-dependent, sustained [Ca2+]i transient, triggered intracellular Ca2+ waves, stimulated Ca2+-ATPase activity, and promoted InsP3 production. The effect of Hg2+ was reduced by the Ca2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca2+-free medium, Hg2+ induced a smaller [Ca2+]i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-betaS, a G protein inhibitor, or heparin, a blocker of intracellular Ca2+ release. Data indicate that Hg2+ induces Ca2+ entry through verapamil-sensitive channels, and intracellular Ca2+ release via a G protein-PLC-InsP3 mechanism. However, in cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+, the [Ca2+]i rise was maximally reduced, indicating that the global effect of Hg2+ is not a mere sum of Ca2+ entry plus Ca2+ release, but involves an amplification of Ca2+ release operated by Ca2+ entry through a CICR mechanism. PMID:12887976

  1. PGC-1α accelerates cytosolic Ca2+ clearance without disturbing Ca2+ homeostasis in cardiac myocytes

    Energy metabolism and Ca2+ handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1α in cardiac Ca2+ signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1α via adenoviral transduction. Our data shows that overexpressing PGC-1α improved myocyte contractility without increasing the amplitude of Ca2+ transients, suggesting that myofilament sensitivity to Ca2+ increased. Interestingly, the decay kinetics of global Ca2+ transients and Ca2+ waves accelerated in PGC-1α-expressing cells, but the decay rate of caffeine-elicited Ca2+ transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), but not Na+/Ca2+ exchange (NCX) contribute to PGC-1α-induced cytosolic Ca2+ clearance. Furthermore, PGC-1α induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1α did not disturb cardiac Ca2+ homeostasis, because SR Ca2+ load and the propensity for Ca2+ waves remained unchanged. These data suggest that PGC-1α can ameliorate cardiac Ca2+ cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1α-calcium handing pathway sheds new light on the role of PGC-1α in the therapy of cardiac diseases.

  2. Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity

    Bhagavathy S; Sumathi P


    Objective: To identify the alteration of the membrane potential and the effect of carotenoid extracts from Chlorococcum humicola (C. humicola) on membrane bound ATPases and lipid peroxidation. Methods: The total carotenoids were extracted from C. humicola. Four groups of Swiss albino mice were treated as control, Benzo(a)pyrene [B(a)P], total carotenoids, B(a)P +total carotenoids respectively for a period of 60 days. Membrane lipid peroxidation and ATPases (Total ATPases, Ca2+- ATPases, Mg2+ - ATPases, Na+K+ - ATPase) were determined in lung, liver and erythrocyte samples. Results: The activity of total ATPase was found to be significantly increased in the B(a)P treated liver and lung tissue. Erythrocyte membrane also showed higher ATPase activity which was significantly reverted on total carotenoid treatment. Conclusions:It can be concluded that the changes in membrane potential favour the functional deterioration of physiological system. The overall findings demonstrates that the animals post treated with carotenoid extract from C. humicola may maintains the alterations in membrane bound ATPase and lipid peroxidation in tissues against the carcinogenic chemical and hence aid in establishing the membrane potential action. Therefore C. humicola can be further extended to exploits its possible application for various health benefits as neutraceuticals and food additives.

  3. Multifaceted plasma membrane Ca(2+) pumps: From structure to intracellular Ca(2+) handling and cancer.

    Padányi, Rita; Pászty, Katalin; Hegedűs, Luca; Varga, Karolina; Papp, Béla; Penniston, John T; Enyedi, Ágnes


    Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate . Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26707182

  4. Substantial depletion of the intracellular Ca2+ stores is required for macroscopic activation of the Ca2+ release-activated Ca2+ current in rat basophilic leukaemia cells.

    Fierro, L; Parekh, A B


    1. Tight-seal whole-cell patch clamp experiments were performed to examine the ability of different intracellular Ca2+ mobilising agents to activate the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL-1) cells under conditions of weak cytoplasmic Ca2+ buffering. 2. Dialysis with a maximal concentration of inositol 1,4,5-trisphosphate (IP3) routinely failed to activate macroscopic ICRAC in low buffer (0.mM EGTA, BAPTA or dimethyl BAPTA), whereas it activated the current to its maximal extent in high buffer (10 mM EGTA). Dialysis with a poorly metabolisable analogue of IP3, with ionomycin, or with IP3 and ionomycin all failed to generate macroscopic ICRAC in low Ca2+ buffering conditions. 3. Dialysis with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin was able to activate ICRAC even in the presence of low cytoplasmic Ca2+ buffering, albeit at a slow rate. Exposure to IP3 together with the SERCA blockers thapsigargin, thapsigargicin or cyclopiazonic acid rapidly activated ICRAC in low buffer. 4. Following activation of ICRAC by intracellular dialysis with IP3 and thapsigargin in low buffer, the current was very selective for Ca2+ (apparent KD of 1 mM) Sr2+ and Ba2+ were less effective charge carriers and Na+ was not conducted to any appreciable extent. The ionic selectivity of ICRAC was very similar in low or high intracellular Ca2+ buffer. 5. Fast Ca2+-dependent inactivation of ICRAC occurred at a similar rate and to a similar extent in low or high Ca2+ buffer. Ca2+-dependent inactivation is not the reason why macroscopic ICRAC cannot be seen under conditions of low cytoplasmic Ca2+ buffering. 6. ICRAC could be activated by combining IP3 with thapsigargin, even in the presence of 100 microM Ca2+ and the absence of any exogenous Ca2+ chelator, where ATP and glutamate represented the only Ca2+ buffers in the pipette solution. 7. Our results suggest that a threshold exists within the IP3-sensitive Ca2+ store

  5. Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

    Reno C Reyes


    Full Text Available Astroglial excitability operates through increases in Ca2+cyt (cytosolic Ca2+, which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na+cyt (cytosolic Na+ control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca2+-ATPase, NCX (plasma membrane Na+/Ca2+ exchanger and NKA (Na+/K+-ATPase in complex and coordinated regulation of Ca2+cyt and Na+cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na+ and Ca2+ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca2+ and Na+ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca2+ and Na+ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca2+-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca2+-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca2+ release from the ER (endoplasmic reticulum.

  6. Effect of Ca2+ on the dimeric structure of scallop sarcoplasmic reticulum


    Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze- fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+- ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isol...

  7. Kinetics of Ca2+ carrier in rat liver mitochondria.

    Bragadin, M; Pozzan, T; Azzone, G F


    The rate of aerobic Ca2+ transport is limited by the rate of the H+ pump rather than by the Ca2+ carrier. The kinetics of the Ca2+ carrier has therefore been studied by using the K+ diffusion potential as the driving force. The apparent Vmax of the Ca2+ carrier is, at 20 degrees C, about 900 nmol (mg of protein)-1 min-1, more than twice the rate of the H+ pump. The apparent Vmax is depressed by Mg2+ and Li+. This supports the view that the electrolytes act as noncompetitive inhibitors of the Ca2+ carrier. The degree of sigmoidicity of the kinetics of Ca2+ transport increases with the lowering of the temperature and proportionally with the concentration of impermeant electrolytes such as Mg2+ and Li+ but not choline. The effects of temperature and of electrolyte do not support the view that the sigmoidicity is due to modifications of the surface potential. Rather, they suggest that Ca2+ transport occurs through a multisubunit carrier, where cooperative phenomena are the result of ligand-induced conformational changes due to the interaction of several allosteric effectors with the carrier subunits. In contrast with La3+ which acts as a competitive inhibitor, Ruthenium Red affects the kinetics by inducing phenomena both of positive and of negative cooperativity. The Ruthenium Red induced kinetics has been reproduced through curve-fitting procedures by applying the Koshland sequential interaction hypothesis to a four-subunit Ca2+ carrier model. PMID:42437

  8. Dietary calcium attenuates platelet aggregation and intracellular Ca2+ mobilization in spontaneously hypertensive rats

    Otsuka, K.; Watanabe, M.; Yue, Q.; McCarron, D. A.; Hatton, D.


    Spontaneously hypertensive rats (SHR) are known to be blood pressure sensitive to dietary calcium. The effects of dietary calcium on platelet aggregation and intracellular Ca2+ mobilization were assessed by turbidimetric methods and fura-2 methods, respectively, in washed platelets of SHR. Ca2+ ATPase activity was examined in aortic membrane fractions. Six weeks of dietary calcium supplementation attenuated the increase of systolic blood pressure (SBP 199 +/- 16 v 170 +/- 9 mm Hg, P ionomycin-induced intracellular calcium ([Ca2+]i) peak in the absence of external Ca2+, which reflects [Ca2+]i storage size, and thrombin-evoked [Ca2+]i release from [Ca2+]i storage were decreased by 2.0% Ca diet (472 +/- 55 v 370 +/- 23 nmol/L, P ionomycin-induced [Ca2+]i (r = 0.591, P = .0415), respectively. However, there was no significant effect of dietary calcium on Ca2+-ATPase activity in aortic membranes. These results suggest that dietary calcium supplementation had a beneficial effect on platelets of SHR by attenuating [Ca2+]i mobilization from [Ca2+]i storage. The hypotensive effect of dietary calcium might be associated with attenuated [Ca2+]i mobilization in SHR.

  9. Cellular mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin-induced rat ventricular hypertrophy

    Lai-jing SONG; Guan-lei WANG; Jie LIU; Qin-ying QIU; Jing-hua OU; Yong-yuan GUAN


    Aim:To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods:Echocardiography was used to confirm the establishment of the cardiac hypertro-phy model. The confocal microscopy and fluorescent indicator Fluo-3 was ap-plied to examine the intracellular Ca2+ concentration ([Ca2+]I), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results:L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]I increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion:The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

  10. State-dependent firing determines intrinsic dendritic Ca2+ signaling in thalamocortical neurons.

    Errington, Adam C; Renger, John J; Uebele, Victor N; Crunelli, Vincenzo


    Activity-dependent dendritic Ca(2+) signals play a critical role in multiple forms of nonlinear cellular output and plasticity. In thalamocortical neurons, despite the well established spatial separation of sensory and cortical inputs onto proximal and distal dendrites, respectively, little is known about the spatiotemporal dynamics of intrinsic dendritic Ca(2+) signaling during the different state-dependent firing patterns that are characteristic of these neurons. Here we demonstrate that T-type Ca(2+) channels are expressed throughout the entire dendritic tree of rat thalamocortical neurons and that they mediate regenerative propagation of low threshold spikes, typical of, but not exclusive to, sleep states, resulting in global dendritic Ca(2+) influx. In contrast, actively backpropagating action potentials, typical of wakefulness, result in smaller Ca(2+) influxes that can temporally summate to produce dendritic Ca(2+) accumulations that are linearly related to firing frequency but spatially confined to proximal dendritic regions. Furthermore, dendritic Ca(2+) transients evoked by both action potentials and low-threshold spikes are shaped by Ca(2+) uptake by sarcoplasmic/endoplasmic reticulum Ca(2+) ATPases but do not rely on Ca(2+)-induced Ca(2+) release. Our data demonstrate that thalamocortical neurons are endowed with intrinsic dendritic Ca(2+) signaling properties that are spatially and temporally modified in a behavioral state-dependent manner and suggest that backpropagating action potentials faithfully inform proximal sensory but not distal corticothalamic synapses of neuronal output, whereas corticothalamic synapses only "detect" Ca(2+) signals associated with low-threshold spikes. PMID:21048143

  11. Modulation of two functionally distinct Ca2+ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger.

    Golovina, V A; Bambrick, L L; Yarowsky, P J; Krueger, B K; Blaustein, M P


    Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, induced a transient, four-fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca(2+)-sensitive stores, induced a two-fold increase in [Ca2+]cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3(CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage-dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller, Ca(2+)- (CAF-) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma. PMID:8721670

  12. Diallyl tetrasulfide improves cadmium induced alterations of acetylcholinesterase, ATPases and oxidative stress in brain of rats

    Cadmium (Cd) is a neurotoxic metal, which induces oxidative stress and membrane disturbances in nerve system. The garlic compound diallyl tetrasulfide (DTS) has the cytoprotective and antioxidant activity against Cd induced toxicity. The present study was carried out to investigate the efficacy of DTS in protecting the Cd induced changes in the activity of acetylcholinesterase (AChE), membrane bound enzymes, lipid peroxidation (LPO) and antioxidant status in the brain of rats. In rats exposed to Cd (3 mg/kg/day subcutaneously) for 3 weeks, a significant (P +K+-ATPase, Mg2+-ATPase and Ca2+-ATPase) were observed in brain tissue. Oral administration of DTS (40 mg/kg/day) with Cd significantly (P < 0.05) diminished the levels of LPO and protein carbonyls and significantly (P < 0.05) increased the activities of ATPases, antioxidant enzymes, GSH and TSH in brain. These results indicate that DTS attenuate the LPO and alteration of antioxidant and membrane bound enzymes in Cd exposed rats, which suggest that DTS protects the brain function from toxic effects of Cd

  13. Effects of mibefradil on intracellular Ca2+ release in cultured rat cardiac fibroblasts and human platelets.

    Eberhard, M; Miyagawa, K; Hermsmeyer, K; Erne, P


    The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 microM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 microM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1 microM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 microM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 microM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+ - nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (> or

  14. Effect of human proinsulin C-peptide on erythrocyte Na+-K+-ATPase activity and Ca2+concentration in patients with type H diabetes mellitus in vitro%胰岛素原C肽对2型糖尿病患者离体红细胞膜Na+-K+-ATP酶活性及红细胞内Ca2+浓度的影响

    李素霞; 袁勤生



  15. Growth-inhibitory effects of a mineralized extract from the red marine algae, Lithothamnion calcareum, on Ca2+-sensitive and Ca2+-resistant human colon carcinoma cells

    Nadeem Aslam, Muhammad; Bhagavathula, Narasimharao; Paruchuri, Tejaswi; Hu, Xin; Chakrabarty, Subhas; Varani, James


    Proliferation and differentiation were assessed in a series of human colon carcinoma cell lines in response to a mineral-rich extract derived from the red marine algae, Lithothamnion calcareum. The extract contains 12% Ca2+, 1% Mg2+, and detectable amounts of 72 trace elements, but essentially no organic material. The red algae extract was as effective as inorganic Ca2+ alone in suppressing growth and inducing differentiation of colon carcinoma cells that are responsive to a physiological level of extracellular Ca2+ (1.4 mM). However, with cells that are resistant to Ca2+ alone, the extract was still able to reduce proliferation and stimulate differentiation. PMID:19394137

  16. Fusion of the Endoplasmic Reticulum and Mitochondrial Outer Membrane in Rats Brown Adipose Tissue: Activation of Thermogenesis by Ca2+

    de Meis, Leopoldo; Ketzer, Luisa A.; da Costa, Rodrigo Madeiro; de Andrade, Ivone Rosa; Benchimol, Marlene


    Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca2+ effect in BAT mitochondria thermogenesis. We found that Ca2+ increased the rate of respiration and heat production ...

  17. Growth-inhibitory effects of a mineralized extract from the red marine algae, Lithothamnion calcareum, on Ca2+-sensitive and Ca2+-resistant human colon carcinoma cells

    Nadeem Aslam, Muhammad; Bhagavathula, Narasimharao; Paruchuri, Tejaswi; Hu, Xin; Chakrabarty, Subhas; Varani, James


    Proliferation and differentiation were assessed in a series of human colon carcinoma cell lines in response to a mineral-rich extract derived from the red marine algae, Lithothamnion calcareum. The extract contains 12% Ca2+, 1% Mg2+, and detectable amounts of 72 trace elements, but essentially no organic material. The red algae extract was as effective as inorganic Ca2+ alone in suppressing growth and inducing differentiation of colon carcinoma cells that are responsive to a physiological lev...

  18. Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores

    Behne, M J; Tu, Chia-Ling L; Aronchik, I; Epstein, E; Bench, G.; Bikle, D D; Pozzan, T; Mauro, T.M.


    Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast and Caenorhabditis elegans. To test whether the ATP2C1 controls Golgi Ca2+ stores, we measu...

  19. 云芝多糖对运动训练大鼠脑组织抗氧化能力和ATPase活性的影响%Effect of Krestin Polysaccharide on Antioxidant Capability and ATPase Activity in Brain Tissues of Rats with High Intensity Exercise

    习雪峰; 王单一; 熊正英; 张林


    目的:探讨云芝多糖对力竭运动大鼠脑组织的部分抗氧化酶活性和Na^2+,K^+-ATP酶(Na^+,K^+.ATPasc),Ca^2+,Mg^2+-ATP酶(Ca^2+,Mg^2+.ATPase)活性影响。方法:选取成年雄性SD大鼠24只。将大鼠随机分为3组:安静对照组8只,运动对照组8只,运动加药组8只,进行为期8周的大强度耐力跑台训练。测定大鼠脑组织部分抗氧化酶和Na^+,K^+-ATPase,Ca^2+,Mg^2+.ATPase活性的变化。结果:与安静对照组相比,运动对照组脑组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)、Na^+,K^+-ATPase、Can,Mg^2+-ATPase活性和血糖含量显著下降(P〈0.01或P〈0.05),MDA生成显著增多(P〈0.05);云芝多糖提高了力竭运动大鼠脑组织SOD、CAT、GSH.Px、T-AOC、Na^+,K^+-ATPase、Ca^2+,Mg^2+.ATPase活性和血糖含量(P〈0.01或P〈0.05),减少MDA生成(P〈0.05)。结论:云芝多糖可以提高力竭运动大鼠脑组织中抗氧化酶活性和Na^+,D.ATPase和Ca^2+,Mg^2+-ATPase的活性,这对于维持运动中能量的供应和抗氧化能力,从而提高大鼠的运动能力具有重要意义。%Objective: To explore the effect of Krestin polysaechatide (PSK) on the activities of antioxidant enzymes and Na^+- K^+-ATPase as well as Ca^2 +-Mg^2+-ATPase in brain tissues of rats after exhaustive exercise. Methods: 24 SD rats were divided into three groups including the control group without exercise, exercise group and exercise plus PSK group. Each group included 8 rats. The rats from the exercise and exercise plus PSK groups were subjected to high-intensity endurance running for 8 consecutive weeks. The changes in the activities of antioxidant enzymes, Na^+-K^+-ATPase and Ca^2+-Mg^2+-ATPase were measured. Results: The activities of SOD

  20. Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

    Brand, M D; Lehninger, A L


    The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP. PMID:1176454

  1. STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.

    Zhao, Guiling; Li, Tianyu; Brochet, Didier X P; Rosenberg, Paul B; Lederer, W J


    In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells. PMID:26261328

  2. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    J.B.R. Rodriguez


    Full Text Available Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endoplasmic reticulum Ca2+-ATPase isoform (SERCA is expressed in rat vas deferens (RVD and its modulation by calmodulin (CaM-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM and a low sensitivity to vanadate (IC50 = 41 µM. These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

  3. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity

  4. Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

    Smith Pete J


    Full Text Available Abstract Background In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport. Results This study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not. Conclusions We have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

  5. Calcium-ATPases: Gene disorders and dysregulation in cancer.

    Dang, Donna; Rao, Rajini


    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26608610

  6. A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus.

    Gonçalves, Rúbia R; Masui, Douglas C; McNamara, John C; Mantelatto, Fernando L M; Garçon, Daniela P; Furriel, Rosa P M; Leone, Francisco A


    To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na

  7. Multiple Ca2+ sensors in secretion

    Walter, Alexander M; Groffen, Alexander J; Sørensen, Jakob Balslev;


    Regulated neurotransmitter secretion depends on Ca(2+) sensors, C2 domain proteins that associate with phospholipids and soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes to trigger release upon Ca(2+) binding. Ca(2+) sensors are thought to prevent spontaneous...... fusion at rest (clamping) and to promote fusion upon Ca(2+) activation. At least eight, often coexpressed, Ca(2+) sensors have been identified in mammals. Accumulating evidence suggests that multiple Ca(2+) sensors interact, rather than work autonomously, to produce the complex secretory response...... observed in neurons and secretory cells. In this review, we present several working models to describe how different sensors might be arranged to mediate synchronous, asynchronous and spontaneous neurotransmitter release. We discuss the scenario that different Ca(2+) sensors typically act on one shared...

  8. Retinoic acid inhibits calmodulin binding to human erythrocyte membranes and reduces membrane Ca2(+)-adenosine triphosphatase activity.

    Davis, F B; Smith, T. J.; Deziel, M R; Davis, P J; Blas, S D


    Ca2(+)-ATPase activity in human red cell membranes is dependent on the presence of calmodulin. All trans-retinoic acid inhibited human red cell membrane Ca2(+)-ATPase activity in vitro in a concentration-dependent manner (10(-8) to 10(-4) M). In contrast, retinol, retinal, 13-cis-retinoic acid and the benzene ring analogue of retinoic acid did not alter enzyme activity. Purified calmodulin (up to 500 ng/ml, 3 X 10(-8) M) added to red cell membranes, in the presence of inhibitory concentration...

  9. When Isolated at Full Receptivity, in Vitro Fertilized Wheat (Triticum aestivum, L. Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin

    Zsolt Pónya


    Full Text Available During in vitro fertilization of wheat (Triticum aestivum, L. in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.

  10. Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

    Glushakova Svetlana


    Full Text Available Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress

  11. SERCA and PMCA pumps contribute to the deregulation of Ca2+ homeostasis in human CF epithelial cells.

    Philippe, Réginald; Antigny, Fabrice; Buscaglia, Paul; Norez, Caroline; Becq, Frédéric; Frieden, Maud; Mignen, Olivier


    Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein. PMID:25661196

  12. Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity.

    Lucena, Malson N; Garçon, Daniela P; Mantelatto, Fernando L M; Pinto, Marcelo R; McNamara, John C; Leone, Francisco A


    We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na(+), K(+))-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg(-1) H(2)O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg(-1) H(2)O). Hemolymph [Na(+)] (323.0 ± 2.5 mmol L(-1)) and [Mg(2+)] (34.6 ± 1.0 mmol L(-1)) are hypo-regulated while [Ca(2+)] (22.5 ± 0.7 mmol L(-1)) is hyper-regulated; [K(+)] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L(-1)) but hypo-regulated (6.2 ± 0.7 mmol L(-1)) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm=46.5 ± 3.5 Umg(-1); K(0.5)=7.07 ± 0.01 μmol L(-1)) and a low-affinity ATP binding site (Vm=108.1 ± 2.5 U mg(-1); K(0.5)=0.11 ± 0.3 mmol L(-1)), both obeying cooperative kinetics, were disclosed. Modulation of (Na(+), K(+))-ATPase activity by Mg(2+), K(+) and NH(4)(+) also exhibits site-site interactions, but modulation by Na(+) shows Michaelis-Menten kinetics. (Na(+), K(+))-ATPase activity is synergistically stimulated up to 45% by NH(4)(+) plus K(+). Enzyme catalytic efficiency for variable [K(+)] and fixed [NH(4)(+)] is 10-fold greater than for variable [NH(4)(+)] and fixed [K(+)]. Ouabain inhibited ≈80% of total ATPase activity (K(I)=464.7 ± 23.2 μmol L(-1)), suggesting that ATPases other than (Na(+), K(+))-ATPase are present. While (Na(+), K(+))-ATPase activities are similar in fresh-caught (around 142 nmol Pi min(-1)mg(-1)) and 45‰-acclimated crabs (around 154 nmol Pi min(-1)mg(-1)), ATP affinity decreases 110-fold and Na(+) and K(+) affinities increase 2-3-fold in 45‰-acclimated crabs. PMID:22260788

  13. Structural and functional studies of heavy metal ATPases

    Sitsel, Oleg


    the bacterial proteins LpCopA and SsZntA, which represent Cu+- and Zn2+-ATPases, respectively. The thesis first compares the recent pioneering P1B-ATPase structure of LpCopA to that of the well-described Ca2+-ATPase SERCA, showing how Cu+-ATPases have managed to adapt the general P-type ATPase...... Zn2+ homeostasis that belong to the superfamily of P-type ATPases, transmembrane proteins which are present in virtually all lifeforms, with functions ranging from membrane potential generation to muscle relaxation. The goal of this thesis is to improve our understanding of P1B-ATPases by focusing on...... crystal structure of LpCopA in a new conformational state is then presented and studied using a variety of methods, showing that Cu+-ATPases use an ion release pathway unique for the P-type ATPase superfamily. The next section introduces the two pioneering crystal structures of a Zn2+-ATPase, Ss...

  14. Cardiomyocyte Ca(2+) dynamics: clinical perspectives.

    Aronsen, Jan Magnus; Louch, William E; Sjaastad, Ivar


    In the heart, Ca(2+) signals regulate a variety of biological functions ranging from contractility to gene expression, cellular hypertrophy and death. In this review, we summarize the role of local Ca(2+) homeostasis in these processes in healthy cardiac muscle cells, and highlight how mismanaged Ca(2+) handling contributes to the pathophysiology of conditions such as cardiac arrhythmia, ischemic heart disease, cardiac hypertrophy and heart failure. Aiming to provide an introduction to the field with a clinical perspective, we also indicate how current and future therapies may modulate cardiomyocytes Ca(2+) handling for the treatment of patients. PMID:26729487

  15. Regulation of free Ca2+ by liver mitochondria and endoplasmic reticulum.

    Becker, G L; Fiskum, G; Lehninger, A L


    Electrode measurements were made of the free Ca2+ concentration maintained by suspensions of isolated rat liver mitochondria and microsomes, as well as by hepatocytes whose plasma membranes had been made permeable by treatment with digitonin. When the KCl, ATP, Mg2+, and phosphate concentrations were made similar to that of cytosol, the steady state free Ca2+ concentration in the presence of respiring mitochondria alone was about 0.5 microM. The additional presence of rat liver microsomes resulted in a steady state level of close to 0.2 microM, which was maintaied for greater than 1 h at 25 degrees C. This concentration of Ca2+ was also maintained by suspensions of hepatocytes permeabilized by digitonin and thus may approximate the actual cytosolic free Ca2+ concentration in vivo. The "set point" for free Ca2+ homeostasis in these systems is determined by mitochondrial Ca2+ influx-efflux cycling, which is dependent on the level of intramitochondrial Ca2+ and can be adjusted by sequestration of Ca2+ in microsomes. PMID:7410406

  16. Capsaicin stimulates the non-store-operated Ca2+ entry but inhibits the store-operated Ca2+ entry in neutrophils

    Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) V1. However, capsaicin-stimulated [Ca2+]i elevation occurred only at high concentrations (≥100 μM). This response was substantially decreased in a Ca2+-free medium. Vanilloids displayed similar patterns of Ca2+ response with the rank order of potency as follows: scutigeral>resiniferatoxin>capsazepine>capsaicin=olvanil>isovelleral. Arachidonyl dopamine (AAD), an endogenous ligand for TRPV1, failed to desensitize the subsequent capsaicin challenge. Capsaicin-induced Ca2+ response was not affected by 8-bromo-cyclic ADP-ribose (8-Br-cADPR), the ryanodine receptor blocker, but was slightly attenuated by 1-[6-[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), the blocker of receptor-gated and store-operated Ca2+ (SOC) channels, 2-aminoethyldiphenyl borate (2-APB), the blocker of D-myo-inositol 1,4,5-trisphospahte (IP3) receptor and Ca2+ influx, and by ruthenium red, a blocker of TRPV channels, and enhanced by the Ca2+ channels blocker, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12330A) and Na+-deprivation. In addition, capsaicin had no effect on the plasma membrane Ca2+-ATPase activity or the production of nitric oxide (NO) and reactive oxygen intermediates (ROI) or on the total thiols content. Capsaicin (≥100 μM) inhibited the cyclopiazonic acid (CPA)-induced store-operated Ca2+ entry (SOCE). In the absence of external Ca2+, the robust Ca2+ entry after subsequent addition of Ca2+ was decreased by capsaicin in CPA-activated cells. Capsaicin alone increased the actin cytoskeleton, and also increased the actin filament content in cell activation with CPA. These results indicate that capsaicin activates a TRPV1-independent non-SOCE pathway in neutrophils. The reorganization of the actin cytoskeleton is

  17. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Narendranath Reddy Chintagari

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  18. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin


    Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H(+) into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+) chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca(2+) release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+) pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+) mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion. PMID:20169059

  19. Downregulation of Ca(2+) and Mg(2+) transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia.

    Nijenhuis, T.; Hoenderop, J.G.J.; Bindels, R.J.M.


    FK506 (tacrolimus) and dexamethasone are potent immunosuppressants known to induce significant side effects on mineral homeostasis, including hypercalciuria and hypomagnesemia. However, the underlying molecular mechanisms remain unknown. The present study investigated the effects of FK506 and dexame

  20. Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

    Kwon, Young Chul; Kim, Sinil; Lee, Yong Seok; Lee, Je Chul; Cho, Myung-Je; Lee, Woo-Kon; Kang, Hyung-Lyun; Song, Jae-Young; Baik, Seung Chul; Ro, Hyeon Su


    HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells. PMID:27095458

  1. Characterization of Mg2+-regulated TRPM7-like current in human atrial myocytes

    Macianskiene Regina


    Full Text Available Abstract Background TRPM7 (Transient Receptor Potential of the Melastatin subfamily proteins are highly expressed in the heart, however, electrophysiological studies, demonstrating and characterizing these channels in human cardiomyocytes, are missing. Methods We have used the patch clamp technique to characterize the biophysical properties of TRPM7 channel in human myocytes isolated from right atria small chunks obtained from 116 patients in sinus rhythm during coronary artery and valvular surgery. Under whole-cell voltage-clamp, with Ca2+ and K+ channels blocked, currents were generated by symmetrical voltage ramp commands to potentials between -120 and +80 mV, from a holding potential of -80 mV. Results We demonstrate that activated native current has dual control by intracellular Mg2+ (free-Mg2+ or ATP-bound form, and shows up- or down-regulation by its low or high levels, respectively, displaying outward rectification in physiological extracellular medium. High extracellular Mg2+ and Ca2+ block the outward current, while Gd3+, SpM4+, 2-APB, and carvacrol inhibit both (inward and outward currents. Besides, divalents also permeate the channel, and the efficacy sequence, at 20 mM, was Mg2+>Ni2+>Ca2+>Ba2+>Cd2+ for decreasing outward and Ni2+>Mg2+>Ba2+≥Ca2+>Cd2+ for increasing inward currents. The defined current bears many characteristics of heterologously expressed or native TRPM7 current, and allowed us to propose that current under study is TRPM7-like. However, the time of beginning and time to peak as well steady state magnitude (range from 1.21 to 11.63 pA/pF, ncells/patients = 136/77 of induced TRPM7-like current in atrial myocytes from different patients showed a large variability, while from the same sample of human atria all these parameters were very homogenous. We present new information that TRPM7-like current in human myocytes is less sensitive to Mg2+. In addition, in some myocytes (from 24 out of 77 patients that current

  2. Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats: influence of maternal Mg2+ supplementation.

    Wold, L E; Norby, F L; Hintz, K K; Colligan, P B; Epstein, P N; Ren, J


    Fetal alcohol syndrome (FAS) is often associated with cardiac hypertrophy and impaired ventricular function in a manner similar to postnatal chronic alcohol ingestion. Chronic alcoholism has been shown to lead to hypomagnesemia, and dietary Mg2+ supplementation was shown to ameliorate ethanol- induced cardiovascular dysfunction such as hypertension. However, the role of gestational Mg2+ supplementation on FAS-related cardiac dysfunction is unknown. This study was conducted to examine the influence of gestational dietary Mg2+ supplementation on prenatal ethanol exposure-induced cardiac contractile response at the ventricular myocyte level. Timed-pregnancy female rats were fed from gestation day 2 with liquid diets containing 0.13 g/L Mg2+ supplemented with ethanol (36%) or additional Mg2+ (0.52 g/L), or both. The pups were maintained on standard rat chow through adulthood, and ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix soft-edge system, and intracellular Ca2+ transients were measured as changes in fura-2 fluorescence intensity (Delta FFI). Offspring from all groups displayed similar growth curves. Myocytes from the ethanol group exhibited reduced cell length, enhanced peak shortening (PS), and shortened time to 90% relengthening (TR90) associated with a normal Delta FFI and time to PS (TPS). Mg2+ reverted the prenatal ethanol-induced alteration in PS and maximal velocity of relengthening. However, it shortened TPS and TR90, and altered the Delta FFI, as well as Ca2+ decay rate by itself. Additionally, myocytes from the ethanol group exhibited impaired responsiveness to increased extracellular Ca2+ or stimulating frequency, which were restored by gestational Mg2+ supplementation. These data suggest that although gestational Mg2+ supplementation may be beneficial to certain cardiac contractile dysfunctions in offspring of alcoholic mothers, caution must be taken, as Mg2

  3. Cloning and characterization of a Ca(2+)/H(+) exchanger from the halophyte Salicornia europaea L.

    Zhang, Liquan; Hao, Jinfeng; Bao, Mulan; Hasi, Agula; Niu, Yiding


    The calcium ion (Ca(2+)), which functions as a second messenger, plays an important role in plants' responses to various abiotic stresses, and Ca(2+)/H(+) exchangers (CAXs) are an important part of this process. In this study, we isolated and characterized a putative Ca(2+)/H(+) exchanger gene (SeCAX3) from Salicornia europaea L., a succulent, leafless euhalophyte. The SeCAX3 open reading frame was 1368 bp long and encoded a 455-amino-acid polypeptide that showed 67.9% similarity to AtCAX3. SeCAX3 was expressed in the shoots and roots of S. europaea. Expression of SeCAX3 was up-regulated by Ca(2+), Na(+), sorbitol, Li(+), abscisic acid, and cold treatments in shoots, but down-regulated by Ca(2+), sorbitol, abscisic acid, and cold treatments in roots. When SeCAX3 was transformed into a Ca-sensitive yeast strain, the transformed cells were able to grow in the presence of 200 mM Ca(2+). Furthermore, SeCAX3 conferred drought, salt, and cold tolerance in yeast. Compared with the control strains, the yeast transformants expressing SeCAX3 were able to grow well in the presence of 30 mM Li(+), 150 mM Mg(2+), or 6 mM Ba(2+). These results showed that the expression of SeCAX3 in yeast suppressed its Ca(2+) hypersensitivity and conferred tolerance to Mg(2+) and Ba(2+). Together, these findings suggest that SeCAX3 might be a Ca(2+) transporter that plays a role in regulating cation tolerance and the responses of S. europaea to various abiotic stresses. PMID:26332662

  4. Involvement of Ca(2+)-Dependent Hyperpolarization in Sleep Duration in Mammals.

    Tatsuki, Fumiya; Sunagawa, Genshiro A; Shi, Shoi; Susaki, Etsuo A; Yukinaga, Hiroko; Perrin, Dimitri; Sumiyama, Kenta; Ukai-Tadenuma, Maki; Fujishima, Hiroshi; Ohno, Rei-ichiro; Tone, Daisuke; Ode, Koji L; Matsumoto, Katsuhiko; Ueda, Hiroki R


    The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals. PMID:26996081

  5. Interleukin-1β activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

    Ghosh, Biswarup; Green, Matthew V; Krogh, Kelly A; Thayer, Stanley A


    The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1β accelerated Ca(2+) clearance in a manner blocked by an IL-1β receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1β activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons. PMID:26843596

  6. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    Villalobo, A; Lehninger, A L


    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered. PMID:6766937

  7. The Relative Contribution of NMDARs to Excitatory Postsynaptic Currents is Controlled by Ca2+-Induced Inactivation

    Valiullina, Fliza; Zakharova, Yulia; Mukhtarov, Marat; Draguhn, Andreas; Burnashev, Nail; Rozov, Andrei


    NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons. PMID:26858606

  8. The Relative Contribution of NMDARs to Excitatory Postsynaptic Currents is Controlled by Ca(2+)-Induced Inactivation.

    Valiullina, Fliza; Zakharova, Yulia; Mukhtarov, Marat; Draguhn, Andreas; Burnashev, Nail; Rozov, Andrei


    NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca(2+). At the same time, they are themselves inhibited by the elevation of intracellular Ca(2+) concentration. It is unclear however, whether the Ca(2+) entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca(2+) buffers. Loading of pyramidal cells with exogenous Ca(2+) buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca(2+) influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg(2+) concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca(2+) buffer capacity of postsynaptic neurons. PMID:26858606

  9. The relative contribution of NMDARs to excitatory postsynaptic currents is controlled by Ca2+-induced inactivation.

    Fliza eValiullina


    Full Text Available NMDA receptors (NMDARs are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSP and prolonged the time window for action potential generation.Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons.

  10. Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells

    Hughes, A R; Bird, G S; Obie, J F;


    to thapsigargin. In nominally Ca(2+)-free medium, the addition of bradykinin to A431 cells rapidly but transiently increased inositol 1,4,5-trisphosphate and, in parallel fashion, transiently increased cytosolic Ca2+. Unexpectedly, under these experimental conditions, epidermal growth factor elicited......+)-ATPase inhibitor thapsigargin still mobilized Ca2+. Finally, histamine, a less efficacious guanine nucleotide-dependent protein-linked receptor agonist, as well as photolyzed, microinjected, caged inositol 1,4,5-trisphosphate, also mobilized Ca2+ after bradykinin. The results of this study show (i) that...

  11. Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling.

    Delvendahl, Igor; Jablonski, Lukasz; Baade, Carolin; Matveev, Victor; Neher, Erwin; Hallermann, Stefan


    Fast synchronous neurotransmitter release at the presynaptic active zone is triggered by local Ca(2+) signals, which are confined in their spatiotemporal extent by endogenous Ca(2+) buffers. However, it remains elusive how rapid and reliable Ca(2+) signaling can be sustained during repetitive release. Here, we established quantitative two-photon Ca(2+) imaging in cerebellar mossy fiber boutons, which fire at exceptionally high rates. We show that endogenous fixed buffers have a surprisingly low Ca(2+)-binding ratio (∼ 15) and low affinity, whereas mobile buffers have high affinity. Experimentally constrained modeling revealed that the low endogenous buffering promotes fast clearance of Ca(2+) from the active zone during repetitive firing. Measuring Ca(2+) signals at different distances from active zones with ultra-high-resolution confirmed our model predictions. Our results lead to the concept that reduced Ca(2+) buffering enables fast active zone Ca(2+) signaling, suggesting that the strength of endogenous Ca(2+) buffering limits the rate of synchronous synaptic transmission. PMID:26015575

  12. Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

    Fiskum, G; Lehninger, A L


    Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process. PMID:36390

  13. Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.

    Baumgart, Joel P; Zhou, Zhen-Yu; Hara, Masato; Cook, Daniel C; Hoppa, Michael B; Ryan, Timothy A; Hemmings, Hugh C


    Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. PMID:26351670

  14. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis

    Yuan Liu


    Full Text Available Heat shock protein 70 (HSP70 maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA, but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R, thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  15. Obesity induces upregulation of genes involved in myocardial Ca2+ handling

    A.P. Lima-Leopoldo


    Full Text Available Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+ handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c, sarcolemmal Na+/Ca2+ exchanger (NCX, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a, ryanodine receptor (RyR2, and phospholamban (PLB mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control or high-fat diet (obese for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05 but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

  16. [Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria].

    Veklich, T O; Kosterin, S O; Shynlova, O P


    In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process. PMID:12199098

  17. Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib.

    Ramón Coca

    Full Text Available Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+-pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of

  18. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

    Inositol 1,4,5-triphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, they propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, and intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [γ-32P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg2+ > > Mn2+ > Ca2+) for the 3-kinase

  19. Localized Ca2+ uncaging induces Ca2+ release through IP3R in smooth muscle

    Min WANG; Zheng CHEN; Yan XING; Xu ZHANG; Xian-zhi DONG; Guang-ju JI


    Aim: Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle. Methods: Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm × O.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 μmol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. Results: Local uncaging of caged Ca2+ was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments. Conclusion: (1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.

  20. Release of Ca2+ from the Endoplasmic Reticulum Contributes to Ca2+ Signaling in Dictyostelium discoideum

    Wilczynska, Zofia; Happle, Kathrin; Müller-Taubenberger, Annette; Schlatterer, Christina; Malchow, Dieter; Fisher, Paul R.


    Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faste...

  1. Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

    ZENG QingHua; LI XingTing; ZHONG GuoGan; ZHANG WenJie; SUN ChengWen


    Using fura-2-acetoxymethyl eater (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]1) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats.The effect of ET-1 on [Ca2+]1 elevation was abolished in the presence of the ETA receptor blocker BQ123,but was not affected by the ETa receptor blocker BQ788. ET-1-induced an increase in [Ca2+]1, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibltors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an Increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETa receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.

  2. Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes


    Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca2+]i elevation was abolished in the presence of the ETA receptor blocker BQ123, but was not affected by the ETB receptor blocker BQ788. ET-1-induced an increase in [Ca2+]i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.

  3. Reactive oxygen and nitrogen species disturb Ca(2+) oscillations in insulin-secreting MIN6 β-cells.

    Antonucci, Salvatore; Tagliavini, Alessia; Pedersen, Morten Gram


    Disturbances in pulsatile insulin secretion and Ca(2+) oscillations in pancreatic β-cells are early markers of diabetes, but the underlying mechanisms are still incompletely understood. Reactive oxygen/nitrogen species (ROS/RNS) are implicated in reduced β-cell function, and ROS/RNS target several Ca(2+) pumps and channels. Thus, we hypothesized that ROS/RNS could disturb Ca(2+) oscillations and downstream insulin pulsatility. We show that ROS/RNS production by photoactivation of aluminum phthalocyanine chloride (AlClPc) abolish or accelerate Ca(2+) oscillations in the MIN6 β-cell line, depending on the amount of ROS/RNS. Application of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitor thapsigargin modifies the Ca(2+) response to high concentrations of ROS/RNS. Further, thapsigargin produces effects that resemble those elicited by moderate ROS/RNS production. These results indicate that ROS/RNS interfere with endoplasmic reticulum Ca(2+) handling. This idea is supported by theoretical studies using a mathematical model of Ca(2+) handling adapted to MIN6 cells. Our results suggest a putative link between ROS/RNS and disturbed pulsatile insulin secretion. PMID:26732126

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

    Mahmmoud, Yasser A.


    Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase.We tested the hypothesis that curcumin may modulat...

  5. Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2+ activity in the vascular smooth muscle cell line A10.

    Vrecl, Milka; Babnik, Monika; Sepčić, Kristina; Žužek, Monika C; Maček, Peter; Diacci, Uroš; Frangež, Robert


    Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca(2+) indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca(2+) activity [Ca(2+)]i in A10 cells. This effect was absent in a Ca(2+)-free medium, indicating that OlyA/PlyB-induced [Ca(2+)]i increase was dependent on Ca(2+) influx into cells. The increase in [Ca(2+)]i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La(3+), ii) the inhibitor of the sodium-calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca(2+)-ATPase inhibitor thapsigargin reduced the [Ca(2+)]i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K(+) in the medium did not prevent OlyA/PlyB-induced [Ca(2+)]i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca(2+)]i is due to an influx of Ca(2+) through a variety of co-existing plasma membrane Ca(2+)-permeable channels, Ca(2+) entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca(2+) release, altogether leading to disturbed Ca(2+) homeostasis in A10 cells. PMID:26320834

  6. Study of substitution limit, structural, bulk magnetic and electrical properties of Ca2+ substituted magnesium ferrite

    The percentage substitution of large cation like Ca2+ for Mg2+ in MgFe2O4 without altering the cubic symmetry and its effect on structural, bulk magnetic and electrical properties have been investigated for Mg1-xCaxFe2O4 (x=0.0-0.35) spinel ferrite system by means of X-ray diffraction (XRD), magnetization, ac susceptibility and dc resistivity measurements. It is found that a maximum of 23% of Ca2+ can be substituted for Mg2+. The variation of magneton number with x can be explained on the basis of Neel's collinear spin model. The Neel temperature determined through ac susceptibility and dc resistivity measurements agree with those calculated theoretically. The variation of electrical resistivity go hand in hand with the variation of the activation energy with the Ca-content

  7. Selective Na+/Ca2+ exchanger inhibition prevents Ca2+ overload-induced triggered arrhythmias

    Nagy, Norbert; Kormos, Anita; Kohajda, Zsófia; Szebeni, Áron; Szepesi, Judit; Pollesello, Piero; Levijoki, Jouko; Acsai, Károly; Virág, László; Nánási, Péter P; Papp, Julius Gy; Varró, András; Tóth, András


    Background and Purpose Augmented Na+/Ca2+ exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca2+i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca2+i rise in conditions when [Ca2+]i was augmented via activation of the late sodium current (INaL) or inhibition of the Na+/K+ pump. Experimental Approach Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca2+i transients (CaTs) were monitored with a Ca2+-sensitive fluorescent dye, Fluo-4. Key Results Enhanced INaL increased the Ca2+ load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca2+]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca2+ release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion. Conclusions and Implications Selective NCX inhibition – presumably by blocking revINCX (reverse mode NCX current) – is effective against arrhythmogenesis caused by [Na+]i-induced [Ca2+]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca2+i handling, should be considered as a promising anti-arrhythmic therapeutic strategy. PMID:25073832

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

    Mahmmoud, Yasser Ahmed


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

  9. Mitochondrial Ca2+ uptake in skeletal muscle health and disease

    Zhou, Jingsong; Yi, Jianxun


    Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as min...

  10. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

    Krishna Samanta

    Full Text Available Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

  11. Can Endocrine disrupters interfere with Ca2+ homeostasis in invertebrate cells?

    L. Canesi


    Full Text Available A wide range of environmental chemicals have been shown to alter the endocrine system of both wildlife and humans. There is increasing evidence that many of these endocrine disruptors (EDs, in particular estrogenic chemicals, can rapidly affect cellular homeostasis and signaling in mammalian Ca2+ systems. In this work, in vitro and in vivo data are summarised on the effects of different compounds known or suspected as EDs on homeostasis in Ca2+ marine invertebrate, the blue mussel Mytilus spp. Both synthetic estrogens and different EDs (DES, BPA, NP, PCB congeners, etc. rapidly increased sytosolic [Ca2+] in mussel hemosytes, as evaluated by FURA2 single cell fluorescence microscopy. The observed [Ca2+] increase was unaffected by the antiestrogen Tamoxifen and was due to either increased influx or release from Ca2+ intracellular stores, depending on the compound. Moreover, different ED,s including the brominated flame retardant TBBPA (tetrabromo bisphenol A induced a dose-dependent inhibition of the plasma membrane Ca2+ -ATPase (PMCA activity from mussel gills in vitro, this supporting a direct effect on membrane pumps. The in vitro effects of EDs were observed at concentrations generally higher than those of E2. However, in vivo, mussel exposure to environmetal concentrations of Bisphenol A (BPA and of the polybrominated diphenyl ether TBDE-47 resulted in large inhibition of PMCA activity in the digestive gland. The results indicate that, in invertebrate like in mammalian systems, interference with Ca2+ homeostasis may represent a significant mode of action of a variety of EDs.

  12. The impact of extracellular and intracellular Ca2+ on ethanol-induced smooth muscle contraction

    Naciye YAKTUBAY DONDAS; Mahir KAPLAN; Derya KAYA; Ergin SiNGiRiK


    Aim:To evaluate the impact of extracellular and intracellular Ca~(2+) on contractions induced by ethanol in smooth muscle.Methods: Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer.Results: Ethanol (164 mmol/L) produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine (50 and 100 μmol/L), a local anesthetic agent, and hexamethonium (100 and 500 μmol/L), a ganglionic blocking agent, failed to affect these contractions, verapamil (1-50 μmol/L) and nifedipine (1-50 μmol/L), selective blockers of L-type Ca~(2+) channels, significantly inhibited the contractile responses of ethanol. Using a Ca~(2+)-free medium nearly eliminated these contractions in the same tissue. Ryanodine (1-50 μmol/L) and ruthenium red (10-100 μmol/L), selective blockers of intracellular Ca~(2+) channels/ryanodine receptors; cyclopiazonic acid (CPA; 1-10 μmol/L), a selective inhibitor of sarcoplasmic reticulum (SR) Ca~(2+)-ATPase; and caffeine (0.5-5 mmol/L), a depleting agent of intracellular Ca~(2+) stores, significantly inhibited the contractile responses induced by ethanol. In addition, the com-bination of caffeine (5 mmol/L) plus CPA (10 μmol/L), and ryanodine (10 μmol/L) plus CPA (10 μmol/L), caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine (5 mmol/L), ryanodine (10 μmol/L) and CPA(10 μmol/L) eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice.Conclusion: Both extracellular and intracellular Ca~(2+) may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.

  13. Estimation of long-term Ca(2+) loss through outlet flow from an agricultural watershed and the influencing factors.

    Zhang, Wenzhao; Yin, Chunmei; Chen, Chunlan; Chen, Anlei; Xie, Xiaoli; Fu, Xingan; Hou, Haijun; Wei, Wenxue


    Soil Ca(2+) loss from agricultural lands through surface runoff can accelerate soil acidification and render soil degradation, but the characteristics of Ca(2+) loss and influencing factors in watershed scale are unclear. This study was carried out in a watershed with various land uses in a subtropical region of China. The outlet flow was automatically monitored every 5 min all year round, and the water samples were collected twice a year from 2001 to 2011. The concentrations of Ca(2+), Mg(2+), K(+), total nitrogen (TN), and total phosphorus (TP) of water samples were measured. The dynamic losses of the nutrients through the outlet flow were estimated, and the relationships between the nutrient losses and rainfall intensity as well as antecedent soil moisture were investigated. The results showed that great variations of nutrient concentrations and losses appeared during the investigation period. The average concentrations of Ca(2+), Mg(2+), K(+), TN, and TP were 0.43, 0.08, 0.10, 0.19, and 0.003 mmol L(-1), respectively. The average Ca(2+) loss reached 1493.79 mol ha(-1) year(-1) and was several times higher than for Mg(2+), K(+), and TN, about 140 times higher than for TP. Rainfall intensity had remarkable effects on Ca(2+) concentration (P soil moisture and Ca(2+) concentration only when rainfall intensity was less than 50 mm day(-1). In a word, much greater amounts of Ca(2+) were lost from the watershed, and this may be one important contributor to the increasing acidification of acidic soils in subtropical regions. PMID:26898929

  14. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto


    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  15. The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

    Shen, Haitao; Liang, Peng; Qiu, Suhua; Zhang, Bo; Wang, Yongli; Lv, Ping


    Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery. PMID:26924456

  16. Modeling the contributions of Ca2+ flows to spontaneous Ca2+ oscillations and cortical spreading depression-triggered Ca2+ waves in astrocyte networks.

    Bing Li

    Full Text Available Astrocytes participate in brain functions through Ca(2+ signals, including Ca(2+ waves and Ca(2+ oscillations. Currently the mechanisms of Ca(2+ signals in astrocytes are not fully clear. Here, we present a computational model to specify the relative contributions of different Ca(2+ flows between the extracellular space, the cytoplasm and the endoplasmic reticulum of astrocytes to the generation of spontaneous Ca(2+ oscillations (CASs and cortical spreading depression (CSD-triggered Ca(2+ waves (CSDCWs in a one-dimensional astrocyte network. This model shows that CASs depend primarily on Ca(2+ released from internal stores of astrocytes, and CSDCWs depend mainly on voltage-gated Ca(2+ influx. It predicts that voltage-gated Ca(2+ influx is able to generate Ca(2+ waves during the process of CSD even after depleting internal Ca(2+ stores. Furthermore, the model investigates the interactions between CASs and CSDCWs and shows that the pass of CSDCWs suppresses CASs, whereas CASs do not prevent the generation of CSDCWs. This work quantitatively analyzes the generation of astrocytic Ca(2+ signals and indicates different mechanisms underlying CSDCWs and non-CSDCWs. Research on the different types of Ca(2+ signals might help to understand the ways by which astrocytes participate in information processing in brain functions.

  17. Ca2+ Alternans in a Cardiac Myocyte Model that Uses Moment Equations to Represent Heterogeneous Junctional SR Ca2+

    Huertas, Marco A; Smith, Gregory D.; Györke, Sándor


    Multiscale whole-cell models that accurately represent local control of Ca2+-induced Ca2+ release in cardiac myocytes can reproduce high-gain Ca2+ release that is graded with changes in membrane potential. Using a recently introduced formalism that represents heterogeneous local Ca2+ using moment equations, we present a model of cardiac myocyte Ca2+ cycling that exhibits alternating sarcoplasmic reticulum (SR) Ca2+ release when periodically stimulated by depolarizing voltage pulses. The model...

  18. Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

    Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente


    homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu(309)/Glu(329). A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the...

  19. Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges.

    Blair, Robert E; Sombati, Sompong; Churn, Severn B; Delorenzo, Robert J


    Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model. PMID:18495112

  20. Preparation of wheat root plasma membrane vesicles and effect of water stress on 45Ca2+ transport activity

    The wheat roots plasma membrane (PM) vesicles were obtained by sucrose gradient centrifugation. The experiment results shows that the wheat roots of Zhengyin No.1 PM H+-ATPase latent activity was 24%, and PM inside-out vesicle (IOV) accounts for 76%. With -1.0 MPa stress of 24h, PM Ca2+-ATPase activity of both orientation wheat roots were increased. Under normal water condition and PEG stress, 62% and 53% of the enzyme activity was inhibited respectively by EGTA, radioactive calcium-45 transport amount was 22.09 nmol/mg pro and 4.17 nmol/mg pro. respectively with PM-IOV.PEG stress results in a decrease of 45Ca2+ transport amount of wheat roots PM-IOV by 81%

  1. Thermodynamic properties of Mg2Si and Mg2Ge investigated by first principles method

    The lattice dynamics and thermodynamic properties of Mg2Si and Mg2Ge are studied based on the first principles calculations. We obtain the phonon dispersion curves and phonon density of states spectra using the density functional perturbation theory with local density approximations. By employing the quasi-harmonic approximation, we calculate the temperature dependent Helmholtz free energy, bulk modulus, thermal expansion coefficient, specific heat, Debye temperature and overall Grueneisen coefficient. The results are in good agreement with available experimental data and previous theoretical studies. The thermal conductivities of both compounds are then estimated with the Slack's equation. By carefully choosing input parameters, especially the acoustic Debye temperature, we find that the calculated thermal conductivities agree fairly well with the experimental values above 80 K for both compounds. This demonstrates that the lattice thermal conductivity of simple cubic semiconductors may be estimated with satisfactory accuracy by combining the Slack's equation with the necessary thermodynamics parameters derived completely from the first principles calculations.

  2. TRP-Na(+)/Ca(2+) Exchanger Coupling.

    Harper, Alan G S; Sage, Stewart O


    Na(+)/Ca(2+) exchangers (NCXs) have traditionally been viewed principally as a means of Ca(2+) removal from non-excitable cells. However there has recently been increasing interest in the operation of NCXs in reverse mode acting as a means of eliciting Ca(2+) entry into these cells. Reverse mode exchange requires a significant change in the normal resting transmembrane ion gradients and membrane potential, which has been suggested to occur principally via the coupling of NCXs to localised Na(+) entry through non-selective cation channels such as canonical transient receptor potential (TRPC) channels. Here we review evidence for functional or physical coupling of NCXs to non-selective cation channels, and how this affects NCX activity in non-excitable cells. In particular we focus on the potential role of nanojunctions, where the close apposition of plasma and intracellular membranes may help create the conditions needed for the generation of localised rises in Na(+) concentration that would be required to trigger reverse mode exchange. PMID:27161225

  3. Effect of sophoridine on Ca(2+) induced Ca(2+) release during heart failure.

    Hu, S-T; Shen, Y-F; Gong, J-M; Yang, Y-J


    Sophoridine is a type of alkaloid extract derived from the Chinese herb Sophora flavescens Ait (kushen) and possess a variety of pharmacological effects including anti-inflammation, anti-anaphylaxis, anti-cancer, anti-arrhythmic and so on. However, the effect of sophoridine on heart failure has not been known yet. In this study, the effect of sophoridine on heart failure was investigated using Sprague-Dawley (SD) rat model of chronic heart failure. Morphological results showed that in medium and high dose group, myofilaments were arranged orderly and closely, intermyofibrillar lysis disappeared and mitochondria contained tightly packed cristae compared with heart failure group. We investigated the Ca(2+) induced Ca(2+) transients and assessed the expression of ryanodine receptor (RyR2) and L-type Ca(2+) channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca(2+) transients were markedly increased in amplitude in medium (deltaF/F(0)=43.33+/-1.92) and high dose groups (deltaF/F(0)=47.21+/-1.25) compared with heart failure group (deltaF/F(0)=16.7+/-1.29, P<0.01), Moreover, we demonstrated that the expression of cardiac DHPR was significantly increased in medium- and high dose-group compared with heart failure rats. Our results suggest that sophoridine could improve heart failure by ameliorating cardiac Ca(2+) induced Ca(2+) transients, and that this amelioration is associated with upregulation of DHPR. PMID:26596316

  4. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.

    Anderson, K; Lai, F A; Liu, Q Y; Rousseau, E; Erickson, H P; Meissner, G


    Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle. PMID:2463249

  5. Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca2+ transport proteins and the effect of taurine on myocardial protection in rabbits

    黄先玫; 朱卫华; 康曼丽


    To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca2+ transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes (Myo[Ca2+]i), activity of SR Ca2+-ATPase(SERCA2a), level of SERCA2a mRNA and Ca2+ released channels(RYR2)mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca2+-ATPase and level of SERCA2a mRNA decreased , while Myo[Ca2+]i increased in DOX-treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[Ca2+]i and the decrease of SERCA2a mRNA induced by doxorubicin. The results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX-induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.

  6. Na(+)-Ca2+ exchange in cultured rat hepatocytes: Evidence against a role in cytosolic Ca2+ regulation or signaling

    Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ([Ca2+]i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of [Ca2+]i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of [Ca2+]i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates [Ca2+]i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. [Ca2+]i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The [Ca2+]i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in [Ca2+]i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of [Ca2+]i in hepatocytes

  7. Nitric oxide inhibits capacitative Ca2+ entry by suppression of mitochondrial Ca2+ handling

    Thyagarajan, Baskaran; Malli, Roland; Schmidt, Kurt; Graier, Wolfgang F; Groschner, Klaus


    Nitric oxide (NO) is a key modulator of cellular Ca2+ signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca2+ entry (CCE) into HEK293 cells by impairment of mitochondrial Ca2+ handling. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 μM, respectively. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 μM), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 μM) or 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 μM) failed to antagonize the inhibitory action of NO on CCE. DEANO (1–10 μM) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca2+ levels, respectively, indicated that DEANO (10 μM) depolarized mitochondria and suppressed mitochondrial Ca2+ sequestration. The inhibitory effect of DEANO on Ca2+ uptake into mitochondria was confirmed by recording mitochondrial Ca2+ during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. DEANO (10 μM) failed to inhibit Ba2+ entry into TG-stimulated cells when extracellular Ca2+ was buffered below 1 μM, while clear inhibition of Ba2+ entry into store depleted cells was observed when extracellular Ca2+ levels were above 10 μM. Moreover, buffering of intracellular Ca2+ by use of N,N′-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl

  8. Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes


    Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 micro...

  9. P-type ATPases.

    Palmgren, Michael G; Nissen, Poul


    P-type ATPases form a large superfamily of cation and lipid pumps. They are remarkably simple with only a single catalytic subunit and carry out large domain motions during transport. The atomic structure of P-type ATPases in different conformations, together with ample mutagenesis evidence, has provided detailed insights into the pumping mechanism by these biological nanomachines. Phylogenetically, P-type ATPases are divided into five subfamilies, P1-P5. These subfamilies differ with respect to transported ligands and the way they are regulated. PMID:21351879

  10. Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

    Ludmila A. Frank


    Full Text Available Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

  11. Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.

    de Meis, Leopoldo; Ketzer, Luisa A; da Costa, Rodrigo Madeiro; de Andrade, Ivone Rosa; Benchimol, Marlene


    Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver. PMID:20209153

  12. Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties

    Quamme Gary A


    Full Text Available Abstract Background Intracellular magnesium is abundant, highly regulated and plays an important role in biochemical functions. Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. We have shown that epithelial magnesium conservation is controlled, in part, by differential gene expression leading to regulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated by magnesium. Results Oligonucleotide microarray analysis was used to identify a novel human gene that encodes a protein involved with Mg2+-evoked transport. We have designated this magnesium transporter (MagT1 protein. MagT1 is a novel protein with no amino acid sequence identity to other known transporters. The corresponding cDNA comprises an open reading frame of 1005 base pairs encoding a protein of 335 amino acids. It possesses five putative transmembrane (TM regions with a cleavage site, a N-glycosylation site, and a number of phosphorylation sites. Based on Northern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. When expressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does not display any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cations did not evoke currents. Large external concentrations of some cations inhibited Mg2+ transport (Ni2+, Zn2+, Mn2+ in MagT1-expressing oocytes. Ca2+and Fe2+ were without effect. Real-time reverse transcription polymerase chain reaction and Western blot analysis using a specific antibody demonstrated that MagT1 mRNA and protein is increased by about 2.1-fold and 32%, respectively, in kidney epithelial cells cultured in low magnesium media relative to normal media and in kidney cortex of mice maintained on low magnesium diets

  13. Spectral components of cytosolic [Ca2+] spiking in neurons

    Kardos, J; Szilágyi, N; Juhász, G;


    We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved into evolutio......We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved into...

  14. P4-ATPases

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg;


    Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases......) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...... include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell. © 2013 The Author(s)....

  15. Arabidopsis transcriptional response to extracellular Ca2þ depletion involves a transient rise in cytosolic Ca2þ

    Zhi Qi


    Ecological evidence indicates a worldwide trend of dramatical y decreased soil Ca2þ levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cel ular mechanism of plants’ responses to Ca2þ depletion. In this study, transcriptional profiling analysis helped identify multiple extracel ular Ca2þ ([Ca2þ]ext) depletion‐respon-sive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2þ ([Ca2þ]cyt) sensors were significantly upregulated, implying that [Ca2þ]cyt has a role in sensing [Ca2þ]ext depletion. Consistent with this observation, [Ca2þ]ext depletion stimulated a transient rise in [Ca2þ]cyt that was negatively influenced by [Kþ]ext, suggesting the involvement of a membrane potential‐sensitive component. The [Ca2þ]cyt response to [Ca2þ]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor‐mediated signaling event. The response was insensi-tive to an animal Ca2þ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3þ, an inhibitor of Ca2þ channels, suppressed the [Ca2þ]ext‐triggered rise in [Ca2þ]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2þ]cyt plays an important role in the putative receptor‐mediated cel ular and transcriptional response to [Ca2þ]ext depletion of plant cel s.

  16. Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture.

    Yamada, Hiroshi; Hayashi, Mitsuko; Uehara, Shunsuke; Kinoshita, Mika; Muroyama, Akiko; Watanabe, Masami; Takei, Koji; Moriyama, Yoshinori


    5-hydroxytryptamine (5-HT) is a precursor and a putative modulator for melatonin synthesis in mammalian pinealocytes. 5-HT is present in organelles distinct from l-glutamate-containing synaptic-like microvesicles as well as in the cytoplasm of pinealocytes, and is secreted upon stimulation by norepinephrine (NE) to enhance serotonin N-acetyltransferase activity via the 5-HT2 receptor. However, the mechanism underlying the secretion of 5-HT from pinealocytes is unknown. In this study, we show that NE-evoked release of 5-HT is largely dependent on Ca2+ in rat pinealocytes in culture. Omission of Ca2+ from the medium and incubation of pineal cells with EGTA-tetraacetoxymethyl-ester inhibited by 59 and 97% the NE-evoked 5-HT release, respectively. Phenylephrine also triggered the Ca2+-dependent release of 5-HT, which was blocked by phentolamine, an alpha antagonist, but not by propranolol, a beta antagonist. Botulinum neurotoxin type E cleaved 25 kDa synaptosomal-associated protein and inhibited by 50% of the NE-evoked 5-HT release. Bafilomycin A1, an inhibitor of vacuolar H+-ATPase, and reserpine and tetrabenazine, inhibitors of vesicular monoamine transporter, all decreased the storage of vesicular 5-HT followed by inhibition of the NE-evoked 5-HT release. Agents that trigger L-glutamte exocytosis such as acetylcholine did not trigger any Ca2+-dependent 5-HT release. Vice versa neither NE nor phenylephrine caused synaptic-like microvesicle-mediated l-glutamate exocytosis. These results indicated that upon stimulation of a adrenoceptors pinealocytes secrete 5-HT through a Ca2+-dependent exocytotic mechanism, which is distinct from the exocytosis of synaptic-like microvesicles. PMID:12065661

  17. Interference with Ca2+ release activated Ca2+ (CRAC) channel function delays T-cell arrest in vivo

    Waite, Janelle C.; Vardhana, Santosh; Shaw, Patrick J.; Jang, Jung-Eun; McCarl, Christie-Ann; Cameron, Thomas O; Feske, Stefan; Dustin, Michael L


    Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca2+]i) elevation. TCR activation triggers increased [Ca2+]i and can arrest T-cell motility in vitro. However the requirement for [Ca2+]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca2+ release-activated Ca2+ (CRAC) channel pathway required for [Ca2+]i elevation in T cells through genetic deletion of stromal interaction molecul...

  18. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase.

    Failer, Bernd U; Braun, Norbert; Zimmermann, Herbert


    We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum. PMID:12167635

  19. Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

    Frandsen Aase


    Full Text Available Abstract Background Ca2+-ATPases of endoplasmic reticulum (SERCAs are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity. Results We show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study. Conclusions These findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

  20. Ca2+-dependent proteolytic activity in crab claw muscle: effects of inhibitors and specificity for myofibrillar proteins

    The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca2+-dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy

  1. Modulation of the matrix redox signaling by mitochondrial Ca2+

    Santo-Domingo, Jaime; Wiederkehr, Andreas; De Marchi, Umberto


    Mitochondria sense, shape and integrate signals, and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance, the molecular nature of the proteins involved in mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes ...

  2. The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

    Schlatterer Christina


    Full Text Available Abstract Background cAMP-induced Ca2+-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca2+-stores: acidic stores and the endoplasmic reticulum (ER. The acidic stores may comprise the contractile vacuole network (CV, the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated. Results Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells. Conclusion The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.

  3. Structure and mechanism of Zn2+-transporting P-type ATPases

    Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele; Autzen, Henriette Elisabeth; Andersson, Magnus; Klymchuk, Tetyana; Nielsen, Anna Marie; Rees, Douglas C; Nissen, Poul; Gourdon, Pontus


    , respectively. The structures reveal a similar fold to Cu+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions by the transporter. The E2P...... extracellular release pathway that resemble PII-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase4, 5 (SERCA) and Na+, K+-ATPase6. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.......Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis1. In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (Znt...

  4. Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger

    The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa has been crystallized by the sitting-drop vapour-diffusion method at 293 K. The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca2+-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca2+ to CBD1 results in conformational changes that stimulate exchange to exclude Ca2+ ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca2+-sensor. In order to clarify the structural scaffold for the Ca2+-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca2+-free form was attempted; the structure of the Ca2+-bound form is already available. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa was crystallized by the sitting-drop vapour-diffusion method at 293 K. The crystals belonged to the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 56.99, c = 153.86 Å, β = 120°, and contained one molecule per asymmetric unit (VM = 2.25 Å3 Da−1) with a solvent content of about 55% (VS = 45.57%). Diffraction data were collected within the resolution range 27.72–3.00 Å using an R-AXIS detector and gave a data set with an overall Rmerge of 10.8% and a completeness of 92.8%

  5. Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells.

    Estevez, Ana Y; Roberts, Randolph K; Strange, Kevin


    The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45-50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60-70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 microM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation approximately 8- and approximately 22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ approximately Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first

  6. Acceleration of Ca(2+) repletion in the junctional sarcoplasmic reticulum and alternation of the Ca(2+)-induced Ca(2+)-release mechanism in hypertensive rat (SHR) cardiac muscle.

    Tanaka, Midori; Tameyasu, Tsukasa


    We estimated the time taken for a repletion of the junctional sarcoplasmic reticulum (JSR) Ca(2+) stores from a family of mechanical restitution curves after twitches of various magnitudes in the cardiac muscle of hypertensive rats (SHR), using a method described previously (Tameyasu et al. Jpn J Physiol. 2004;54:209-19), to evaluate abnormality in Ca(2+) handling by cardiac JSR in hypertension. We found no differences in contractility or in the time course of mechanical restitution between SHR and the controls (WKY) at 3 weeks of age. In comparison to WKY, 7- and 20-week-old SHR showed a greater rested state contraction (RST) and similar or smaller rapid cooling contracture, suggesting that their JSR contains a similar amount of Ca(2+) at saturation, but releases more Ca(2+) upon stimulation. The adult SHR and WKY showed similar mechanical restitution time courses, but the adults had longer pretwitch latencies. The function G(t) representing the time course of JSR Ca(2+) store repletion in adult SHR exceeded the WKY value at t JSR [Ca(2+)] change corresponding to the mechanical restitution after RST was smaller in the adult SHR at t JSR Ca(2+) store repletion and an alternation of the Ca(2+)-induced release of Ca(2+ )from the JSR in young adult SHR. PMID:18312741

  7. General requirement for harvesting antennae at Ca2+ and H+ channels and transporters

    Cristián Martínez


    Full Text Available The production and dissipation of energy in cells is intimately linked to the movement of small molecules in and out of enzymes, channels and transporters. An analytical model of diffusion was described previously, which was used to estimate local effects of these proteins acting as molecular sources. The present article describes a simple but more general model, which can be used to estimate the local impact of proteins acting as molecular sinks. The results show that the enzymes, transporters and channels, whose substrates are present at relatively high concentrations like ATP, Na+, glucose, lactate and pyruvate, do not operate fast enough to deplete their vicinity to a meaningful extent, supporting the notion that for these molecules the cytosol is a well-mixed compartment. One specific consequence of this analysis is that the well-documented cross-talk existing between the Na+/K+ ATPase and the glycolytic machinery should not be explained by putative changes in local ATP concentration. In contrast, Ca2+ and H+ transporters like the Na+/Ca2+ exchanger NCX and the Na+/H+ exchanger NHE, show experimental rates of transport that are two to three orders of magnitude faster than the rates at which the aqueous phase may possibly feed their binding sites. This paradoxical result implies that Ca2+ and H+ transporters do not extract their substrates directly from the bulk cytosol, but from an intermediate “harvesting” compartment located between the aqueous phase and the transport site.

  8. Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.

    Meissner, G; Rousseau, E; Lai, F A


    The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel. PMID:2536370

  9. Optical properties and electronic band structure of BiMg2PO6, BiMg2VO6, BiMg2VO6:Pr3+ and BiMg2VO6:Eu3+

    Barros, A.; Deloncle, R.; Deschamp, J.; Boutinaud, P.; Chadeyron, G.; Mahiou, R.; Cavalli, E.; Brik, M. G.


    The luminescence properties of the yellow pigment BiMg2VO6 are revisited and those of BiMg2PO6, BiMg2VO6:Pr3+ and BiMg2VO6:Eu3+ are described. It is shown that the undoped systems exhibit broad band emission in the green or orange spectral regions, but only upon UV or near UV excitation. In contradiction with a previous report, we found that the blue, host absorbed, photons are lost non-radiatively and do not contribute to the luminescence processes in BiMg2VO6. To understand these experimental results, the optical properties of BiMg2VO6 and BiMg2PO6 are theoretically analysed on the basis of electronic structure diagrams calculated by the DFT method. It is found that the optical transitions are mostly localised within [VO4]3- units or non-regular Bi3+ ions and occur in the UV or near UV regions. The luminescence of the trivalent lanthanide dopants is weak (Eu3+) or unobserved (Pr3+) in BiMg2VO6 which is explained by inefficient energy migration in the host lattice to the impurity sites.

  10. Different response of osteoblastic cells to Mg(2+), Zn(2+) and Sr(2+) doped calcium silicate coatings.

    Hu, Dandan; Li, Kai; Xie, Youtao; Pan, Houhua; Zhao, Jun; Huang, Liping; Zheng, Xuebin


    Mg(2+), Zn(2+) and Sr(2+) substitution for Ca(2+) in plasma sprayed calcium silicate (Ca-Si) coatings have been reported to impede their degradation in physiological environment and, more importantly, to improve their biological performance. The reason for the improved biological performance is still elusive and, especially, the contribution of the dopant ions is lack of obvious and direct evidence. In this study, we aim to identify the effect of Mg(2+), Zn(2+) and Sr(2+) incorporation on the osteogenic ability of Ca-Si based coatings (Ca2MgSi2O7, Ca2ZnSi2O7 and Sr-CaSiO3) by minimizing the influence of Ca and Si ions release and surface physical properties. Similar surface morphology, crystallinity and roughness were achieved for all samples by optimizing the spray parameters. As expected, Ca and Si ions release from all the coatings showed the comparable concentration with immersing time. The response of MC3T3-E1 cells onto Mg(2+), Zn(2+) and Sr(2+) doped Ca-Si coatings were studied in terms of osteoblastic adhesion, proliferation, differentiation and mineralization. The results showed that the level of cell adhesion and proliferation increased the most on the surface of Mg-modified coating. Gene expressions of early markers of osteoblast differentiation (COL-I and ALP mRNA) were obviously improved on Zn-modified coating. Gene expressions of later markers for osteoblast differentiation (OPN and OC mRNA) and mineralized nodules formation were obviously accelerated on the surface of Sr-modified coating. Since Mg(2+), Zn(2+) and Sr(2+) play a regulatory role in different stages of osteogenesis, it may be possible to utilize this in the development of new coating materials for orthopedic application. PMID:26787488