Stimulatory effect of regucalcin on mitochondrial ATP-dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca(2+) signaling, on Ca(2+)-ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-6) M) or lanthunum chloride (10(-6) M), an inhibitor of mitochondrial Ca(2+) uptake, markedly inhibited regucalcin (100 nM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (100 nM) in elevating Ca(2+)-ATPase activity was completely prevented by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca(2+)-ATPase activity was not further enhanced by calmodulin (0.30 microM) or dibutyryl cyclic AMP (10(-4) M), which could increase Ca(2+)-ATPase activity. Trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, significantly decreased Ca(2+)-ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca(2+)-pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase activity in the nuclei of regenerating rat liver

The effect of Ca²⁺-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca²⁺ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10⁻⁴ M), or staurosporine (10⁻⁷ M), indicating that Ca²⁺-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10⁻⁷ M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca²⁺/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca²⁺ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca²⁺ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca²⁺ added. Meanwhile, Cd² (25–100 µM)-induced decrease in Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca²⁺ in liver cells.     

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca²⁺/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca²⁺ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca²⁺ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca²⁺/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Regucalcin increases Ca2+-ATPase activity in the mitochondria of brain tissues of normal and transgenic rats

The role of regucalcin, which is a regulatory protein in intracellular signaling, in the regulation of Ca(2+)-ATPase activity in the mitochondria of brain tissues was investigated. The addition of regucalcin (10(-10) to 10(-8) M), which is a physiologic concentration in rat brain tissues, into the enzyme reaction mixture containing 25 microM calcium chloride caused a significant increase in Ca(2+)-ATPase activity, while it did not significantly change in Mg(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca(2+)-ATPase activity was completely inhibited in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), both of which are inhibitors of mitochondrial uniporter activity. Whether the effect of regucalcin is modulated in the presence of calmodulin or dibutyryl cyclic AMP (DcAMP) was examined. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 microg/ml) which significantly increased the enzyme activity. DcAMP (10(-6) to 10(-4) M) did not have a significant effect on Ca(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not seen in the presence of DcAMP (10(-4) M). Regucalcin levels were significantly increased in the brain tissues or the mitochondria obtained from regucalcin transgenic (RC TG) rats. The mitochondrial Ca(2+)-ATPase activity was significantly increased in RC TG rats as compared with that of wild-type rats. This study demonstrates that regucalcin has a role in the regulation of Ca(2+)-ATPase activity in the brain mitochondria of rats.     

Modulation of ATPase activities in the central nervous system by the S-100 proteins

The isomeric forms of bovine S-100a and S-100b have been shown to stimulate ATPase activities in fractions enriched in myelin and mitochondria isolated from the Gerbil brain and for S-100b more effectively than for calmodulin in erythrocytes or skeletal muscle. In the presence of Ca²⁺, S-100a produced a slight increase of ATPase activity in the mitochondrial fraction. However, S-100b, with or without Ca²⁺ and Zn²⁺ respectively, had no effect on the ATPase activity in mitochondria of the Gerbil liver. The observations may indicate a second messenger role for S-100b in the presence of Zn²⁺ in the Schwann cell.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

The effects of felodipine and bepridil on calcium-stimulated calmodulin binding and calcium pumping ATPase of cardiac sarcolemma before and after removal of endogenous calmodulin

Summary The Ca²⁺ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca²⁺-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca²⁺ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca²⁺ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca²⁺ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca²⁺ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca²⁺ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca²⁺-stimulated binding of calmodulin.Abbreviations Ca²⁺ pumping ATPase Ca²⁺ stimulated Mg²⁺-dependent ATP hydrolyzing activity - Na⁺ pumping ATPase Na⁺-stimulated K⁺- and Mg²⁺-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid     

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca2+-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10-8 to 10-6 M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 × 10-6%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 × 10-7 M) to increase Ca2+-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca2+-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10-5 and 10-3 M), while inositol 1,4,5-trisphosphate (10-7 and 10-5 M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10-7 and 10-6 M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10-4 and 10-3 M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca2+-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (Hr-II-E) is stimulated through Ca2+ signaling factors: Involvement of protein kinase C

The expression of hepatic Ca²⁺-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10^-6 M), a Ca²⁺ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10^-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10^-6 M) or estrogen (10^-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10^-9 M) or dexamethasone (10^-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10^-5 M), an antagonist of calmodulin, or staurosporine (10^-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca²⁺-dependent signaling factors.     

Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver

To examine ryanodine‐sensitive Ca²⁺ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α‐ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage‐sensitive fluorescent probe tetramethylrhodamine‐methyl‐ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m‐chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo‐4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α‐ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca²⁺ concentration in the mitochondria matrix under oxidation of pyruvate or α‐ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra‐mitochondrial Ca²⁺ concentration and affecting the energy state of mictochondria in hepatocytes.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

Acute inhibitory effect of alpha‐mangostin on sarcoplasmic reticulum calcium‐ATPase and myocardial relaxation

The benefits of α‐mangostin for various tissues have been reported, but its effect on the heart has not been clarified. This study aimed to evaluate the effects of α‐mangostin on cardiac function. Using a cardiac sarcoplasmic reticulum (SR) membrane preparation, α‐mangostin inhibited SR Ca²⁺‐ATPase activity in a dose‐dependent manner (IC₅₀ of 6.47 ± 0.7 μM). Its suppressive effect was specific to SR Ca²⁺‐ATPase but not to myofibrillar Ca²⁺‐ATPase. Using isolated cardiomyocytes, 50 μM of α‐mangostin significantly increased the duration of cell relengthening and increased the duration of Ca²⁺ transient decay, suggesting altered myocyte relaxation. The relaxation effect of α‐mangostin was also supported in vivo after intravenous infusion. A significant suppression of both peak pressure and rate of ventricular relaxation (–dP/dt) relative to DMSO infusion was observed. The results from the present study demonstrated that α‐mangostin exerts specific inhibitory action on SR Ca²⁺‐ATPase activity, leading to myocardial relaxation dysfunction.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.