Suppressive role of endogenous regucalcin in the regulation of nitric oxide synthase activity in heart muscle cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of nitric oxide (NO) synthase activity in the cytosol of rat heart muscle was investigated. The addition of calcium chloride (5-20 microM) into the enzyme reaction mixture containing the heart cytosolic protein caused a significant increase in NO synthase activity. The Ca2+ effect was significantly inhibited by trifluoperazine (TFP; 20 or 50 microM), an antagonist of calmodulin, indicating the existence of Ca2+/calmodulin-dependent NO synthase activity in rat heart muscle cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-9) or 10(-8) M). This effect was also seen in the presence of calcium chloride (10 microM), TFP (50 microM) or EGTA (1 mM), a chelator of Ca2+. Meanwhile, the effect of regucalcin (10(-8) M) in decreasing NO synthase activity was not seen in the presence of Nw-nitro-L-arginine methylester (NAME; 10(-6) or 10(-5) M), an inhibitor of the enzyme. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This effect was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the heart muscle cytosol of transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium (10 micro M) addition in increasing NO synthase activity was significantly weakened in the heart muscle cytosol of regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the heart muscle cytosol of rats.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.

The effect of Ca2+-binding protein regucalcin on neutral phosphatase activity in rat brain cytosol was investigated. Phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The presence of calcium chloride (10(-5) and 10(-4) M) in the enzyme reaction mixture caused a significant increase in phosphatase activity toward three phosphoaminoacids. The enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (1 or 5 microg/ml) in the presence of calcium (10(-5) M). Such an effect was not seen in the presence of phosphotyrosine. Trifluoperazine (2x10(-5) M), an antagonist of calmodulin, completely inhibited calcium (10(-5) M)-increased phosphatase activity toward phosphoserine and phosphothreonine, whereas it had no effect on the enzyme activity toward phosphotyrosine. Regucalcin (10(-9) M) significantly inhibited phosphatase activity toward three phosphoaminoacids without or with Ca2+ addition. The inhibitory effect of regucalcin (10(-10) and 10(-9) M) was also seen in the presence of Ca2+ (10(-5) M) and calmodulin (5 microg/ml). The presence of anti-regucalcin monoclonal antibody (20 or 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of phosphatase activity toward three phosphoaminoacids; this effect was completely abolished by addition of regucalcin (10(-9) M). The present study suggests that the endogenous regucalcin has an inhibitory effect on Ca2+/calmodulin-dependent protein phosphatase activity in rat brain cytosol.     

Regulatory effect of regucalcin on nitric oxide synthase activity in rat kidney cortex cytosol: Role of endogenous regucalcin in transgenic rats

The effect of regucalcin, a regulatory protein in Ca2+ signaling, on nitric oxid (NO) synthase activity in the cytosol of kidney cortex of rats was investigated. The presence of calcium chloride (10 micro M) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This increase was significantly prevented by the addition of trifluoperazine (TFP; 20 or 50 micro M), an antagonist of calmodulin, supporting the existence of Ca2+/calmodulin-dependent NO synthase in rat kidney cortex cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-10)-10(-8) M) in the reaction mixture in the absence or presence of calcium chloride (10 micro M). The regucalcin (10(-8) M) effect was not seen in the presence of Nw-nitro-L-argine metylester (NAME; 10(-6) or 10(-5) M), an inhibitor of NO synthase. Regucalcin significantly reduced NO synthase activity in the presence of TFP (50 micro micro M) or EGTA (1 mM) which has a significant inhibitory effect on the enzyme activity. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in NO synthase activity. This increase was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the kidney cortex cytosol of regucalcin transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium chloride (10 micro M) in increasing NO synthase activity in the kidney cortex cytosol of wild-type rats was significantly weakened in regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the kidney cortex cytosol of rats.     

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.     

Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: attenuation with increasing age

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 micro M) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 micro M), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10(-4)-10(-8) M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 micro M). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10(-6) M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.     

Enhancement of protein kinase activity in the cytosol of regenerating rat liver: regulatory role of endogenous regucalcin

The effect of regucalcin, a Ca2+-binding protein, on protein kinase activity in the cytosol of regenerating rat liver was investigated. Protein phosphorylation was significantly increased in the liver cytosol obtained at 6, 24, and 48 h after a partial hepatectomy (about 65%) in comparison with that of sham-operated rats. This increase was significantly inhibited by the addition of trifluoperazine (2x10(-5) M), staurosporine (10(-7) M) or genistein (10(-5) M), which is an inhibitor of protein kinases, in the reaction mixture. The presence of regucalcin (0.1-0.5 microM) caused a significant decrease in protein phosphorylation in the cytosol from normal and regenerating rat livers. The effect of regucalcin (0.5 microM) was completely abolished by the addition of anti-regucalcin monoclonal antibody (50 ng/ml). The elevation of protein phosphorylation in regenerating rat liver was significantly enhanced by the presence of anti-regucalcin monoclonal antibody (50 ng/ml). The effect of regucalcin in decreasing protein phosphorylation in the cytosol of regenerating rat liver was not seen in the presence of the antibody. The present study demonstrates that protein kinase activity, is enhanced in the cytosol of regenerating rat liver, and that endogenous regucalcin has an inhibitory role in the enhancement of protein phosphorylation by various protein kinases.     

Brain microsomal calcium accumulation in rats with increasing age: involvement of thapsigargin-sensitive Ca2+-ATPase

The alteration in calcium content and Ca2+-ATPase activity in the brain microsomes of rats with increasing age was investigated. Brain microsomal calcium content was significantly increased in aged rats (50 weeks old) as compared with that of young rats (5 weeks old). Such an increase was not seen in the brain mitochondria of aged rats. Increasing age caused a significant elevation of Ca2+-ATPase activity in the brain microsomes. The microsomal Ca2+-ATPase activity of young rats was significantly decreased by the addition of thapsigargin (10-7-10-5 M), a specific inhibitor of microsomal Ca2+ pump enzyme (Ca2+-ATPase), in the enzyme reaction mixture. The inhibitory effect of thapsigargin (10-5 M) was also seen in the brain microsomes of aged rats. Moreover, the effect of calcium (5 and 10 microM) addition in elevating Ca2+-ATPase activity was not revealed in the microsomes of aged rats, whereas the metal could increase the enzyme activity in young rats, suggesting an involvement of activatory factor on the enzyme with increasing age. The present study demonstrates that ageing induces an increase in thapsigargin-sensitive Ca2+-ATPase activity and a corresponding elevation of calcium content in the brain microsomes. This finding suggests a cellular mechanism by which ageing causes calcium accumulation in brain.     

Role of endogenous regucalcin in transgenic rats: suppression of protein tyrosine phosphatase and ribonucleic acid synthesis activities in liver nucleus

The role of endogenous regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of liver nuclear function was investigated by using regucalcin transgenic (TG) rats. Regucalcin levels were significantly increased in the liver nuclei of regucalcin TG male and female rats. Nuclear protein tyrosine phosphatase activity was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture containing liver nuclear protein obtained from normal (wt) rats. This increase was significantly prevented in the liver nuclei of regucalcin TG rats. Moreover, nuclear ribonucleic acid (RNA) synthesis was significantly suppressed in the liver nuclei of regucalcin TG rats as compared with that of normal rats. The effect of calcium chloride (10 micro M) or anti-regucalcin monoclonal antibody (50 ng/ml) in increasing RNA synthesis was completely blocked in the liver nuclei of the TG rats. These results suggest that endogenous regucalcin plays a suppressive role in the regulation of liver nuclear function in rats.     

Decrease in regucalcin level and enhancement of protein tyrosine phosphatase activity in rat brain microsomes with increasing age

The role of regucalcin, a regulatory protein in Ca2+ signaling, in the microsomes of brain tissue in rats has not been clarified so far. Western blot analysis showed that regucalcin was present in the brain microsomes. Regucalcin levels were significantly decreased in the brain microsomes obtained from 50-week-old rats as compared with that of 5-week-old rats. Meanwhile, protein tyrosine phosphatase activity was seen in the brain microsomes. The enzyme activity was significantly increased with increasing age. The presence of regucalcin (10(-9) M) in the enzyme reaction mixture containing the brain microsomes obtained from 50-week-old rats caused a significant decrease in protein tyrosine phosphatase activity. Such a decrease was not seen in the brain microsomes from 5-week-old rats. Moreover, the presence of anti-regucalcin monoclonal antibody (10 ng/ml) in the enzyme reaction mixture containing the brain microsomes from young and aged rats caused a significant increase in protein tyrosine phosphatase activity, indicating a suppressive role of microsomal endogenous regucalcin. The present study demonstrates that regucalcin is present in the brain microsomes, and that its level is decreased with increasing age. This decrease may be partly involved in the enhancement of protein tyrosine phosphatase activity in the brain mirosomes with increasing age.     

Decrease in Ca2+-ATPase activity in the brain plasma membrane of rats with increasing age: involvement of brain calcium accumulation

The alteration in Ca(2+)-ATPase activity in the brain plasma membrane of rats with increasing age was investigated. Calcium content in the brain tissues was significantly raised in aged rats (50 weeks old) as compared with that of young rats (5 weeks old). Increasing age caused a significant decrease in Ca(2+)-ATPase activity in the brain plasma membranes. The presence of N-ethylmaleimide (2.5 or 5 mM), a modifying reagent of thiol (SH)-groups, in the reaction mixture caused a significant decrease in the brain plasma membrane Ca(2+)-ATPase activity of young and aged rats, while dithiothreitol (2.5 or 5 mM), a protecting reagent of SH-groups, produced a significant increase in the enzyme activity, indicating that the SH-group is an active site of Ca(2+)-ATPase. The active site of Ca(2+)-ATPase may not be impaired by ageing. The brain plasma membrane Ca(2+)-ATPase activity of young rats was significantly reduced in the presence of dibutyryl cyclic AMP (10(-7)-10(-5) M) or inositol 1, 4, 5-trisphosphate (10(-7)-10(-5) M) in the reaction mixture. Such an decrease was not seen in aged rats. The responsibility for signaling factors seemed to be weakened by ageing. Calmodulin (2.5 and 5 microg/ml) or regucalcin (10(-8) and 10(-7) M), a Ca(2+)-regulating protein, did not have an effect on Ca(2+)-ATPase activity. This study demonstrates that ageing induces a decrease in Ca(2+)-ATPase activity in the brain plasma membranes. This finding suggests a cellular mechanism by which ageing causes calcium accumulation in brain.     

Hormonal regulation of regucalcin mRNA expression in osteoblastic MC3T3-E1 cells

The effect of various hormones on regucalcin mRNA expression in osteoblastic MC3T3-E1 cells in vitro was investigated. Cells with subconfluency were cultured for 24 or 48 h in a medium containing either vehicle or various hormones without fetal bovine serum. Regucalcin mRNA expression was significantly increased after culture with parathyroid hormone (synthetic human PTH; 10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M), or 17beta-estradiol (10(-10) or 10(-9) M) for 48 h. Culture with 1,25-dihydroxyvitamin D3 (10(-7) M) for 48 h caused a significant decrease in regucalcin mRNA expression. Regucalcin mRNA expression was significantly decreased after culture with tumor necrosis factor-alpha (1 or 10 ng/ml of medium) for 24 or 48 h. The effect of PTH or IGF-I in increasing regucalcin mRNA expression was not seen in the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or PD98059 (10(-7) M), an inhibitor of mitosis-activated protein kinase (MAP kinase), respectively, suggesting that regucalcin mRNA expression is enhanced through intracellular signaling factors. This study demonstrated that regucalcin mRNA expression in osteoblastic MC3T3-E1 cells is regulated by various hormones.     

Regucalcin increases superoxide dismutase activity in the heart cytosol of normal and regucalcin transgenic rats

The effect of regucalcin, a regulatory protein in the intracellular signaling system, on superoxide dismutase (SOD) activity in the heart cytosol of normal rats and regucalcin transgenic (TG) rats was investigated. The addition of regucalcin (10(-10) to 10(-8) M) with a physiologic concentration in the enzyme reaction mixture containing the heart cytosol obtained from normal rats caused a significant increase in SOD activity, indicating that regucalcin directly activates the enzyme. The effect of regucalcin (10(-8) M) in increasing SOD activity was not seen in the presence of dithiothreitol (DTT; 0.1 or 1.0 mM), a protecting reagent for sulfhydryl group, or N-ethylmaleimide (NEM; 0.1 or 1.0 mM), a modifying reagent for sulfhydryl group, in the reaction mixture, indicating that regucalcin does not affect the sulfhydryl group. The addition of zinc sulfate (10(-6) to 10(-4) M) in the reaction mixture did not cause a significant change in SOD activity, while the enzyme activity was markedly decreased in the presence of cupric sulfate (10(-6) to 10(-4) M). The activatory effect of regucalcin (10(-8) M) on SOD was seen in the presence of zinc (10(-4) M), while not observed in the presence of copper (10(-4) M). Moreover, SOD activity was significantly enhanced in the heart cytosol of regucalcin TG rats as compared with that of normal rats. This study demonstrates that regucalcin increases SOD activity in the heart cytosol of rats, and that its effect is not related to the sulfhydryl group of enzymes.     

缺血等因素对大鼠心肌线粒体Ca^2＋转运的影响

本研究检测了体外缺血等因素对心肌线粒体Ca~(2+)转运的影响。结果表明,缺血明显抑制线粒体Ca~(2+)-ATP酶活性及~(45)Ca摄取率。缺血10分钟时,线粒体Ca~(2+)-ATP酶活性开始下降,40分钟时显著降低,~(45)Ca摄取率的变化也呈类似趋势。去甲肾上腺素及cAMP对线粒体Ca~(2+)-ATP酶活性,~(45)Ca摄取率无任何影响。无机磷对线粒体Ca~(2+)-ATP酶有轻微的抑制作用,二磷酸腺苷对谚酶活性有明显抑制作用,其IC_(50)值为2.5mmol/L。     

A high affinity Ca2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Addition of Ca2+ (0.01-1 mM) to a standard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm-3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked alpha-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca2+, of 90 and 21 mU mg-1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca2+-ATPase, in addition to having no Mg2+ requirement, was inhibited by submicromolar levels of Mg2+. The Ca2+-ATPase was found to have a K0.5 = 128 +/- 22 nM free Ca2+, the response to increasing Ca2+ concentration displaying an extremely high degree of co-operativity (Hill number (nH) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K0.5 = 6.2 +/- 0.61 microM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites (nH = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V2O3-8) having any effect: 47% inhibition at 5.0 microM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (50 microM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca2+ in bloodstream T. rhodesiense.     

新城疫病毒对BGC-823胃癌细胞线粒体的影响

目的 探讨新城疫病毒对BGC-823胃癌细胞线粒体结构和三磷酸腺苷(ATP)酶活性等功能的影响.方法 应用电子显微镜观察、线粒体Na+-K+-ATP酶、Ca2+-ATP酶活性变化测定、罗丹明123染色法测定细胞线粒体膜电位及Western Blot测定细胞色素C等方法 进行分析.结果 新城疫病毒感染肿瘤细胞线粒体结构破坏,线粒体Na+-K+ATP酶、Ca2+-ATP酶活性较对照组显著下降(P<0.01).线粒体膜电位及细胞色素C显著下降(P<0.01).结论 对线粒体结构及功能的影响可能是新城疫病毒杀伤BGC-823胃癌细胞的机制之一.     

The opioid growth factor receptor in human head and neck squamous cell carcinoma

Regucalcin is a Ca2+-binding protein, which plays a regulatory role in liver cell functions related to Ca2+. In this study we have cloned cDNA for regucalcin from rabbit, bovine, chicken and toad livers by using rapid amplification of cDNA ends (RACE) method. The nucleotide and amino acid sequences of them are compared with published human, rat and mouse sequences. Comparison analysis revealed that the nucleotide sequences of regucalcin from seven vertebrate species were highly conserved in their coding region. The overall regucalcin proteins in these species consisted of 299 amino acids, and they had 69.9-91.3% identity. Furthermore, phylogenetic analysis showed that regucalcin in seven species appears to form a single cluster. This study demonstrates a great conservation of the regucalcin genes throughout evolution.     

高血压大鼠红细胞膜钙泵功能障碍机理的初步分析

本工作比较自发性高血压大鼠(SHR)和肾性高血压大鼠(RHR)及对照大鼠红细胞膜Ca~(2+),Mg~(2+)-ATP酶(钙泵)活性及其对CaM、TFP、川芎嗪和硝苯吡啶(Nifedipine、Nif)等的反应,目的是分析高血压时钙泵功能障碍的机理,并寻找能有效提高钙泵活性的药物,为高血压防治提供有效措施。 结果表明SHR及RHR基础钙泵活性明显低于相应对照大鼠钙泵活性;原因之一可能与高血压动物质膜钙泵对钙泵抑制剂TFP的敏感性增加有关,对RHR,川芎嗪作用很类似TFP。提示此药有CaM拮抗作用。Nif除了公认为钙通道阻断剂外,本实验表明对RHR它还有激活钙泵的作用。     

Ruthenium red and magnesium ion partially inhibit silver ion-induced release of calcium from sarcoplasmic reticulum of frog skeletal muscles

Effects of Ca2+-induced Ca2+ release blockers, ruthenium red (RR) and Mg2+, on Ag+-induced Ca2+ release were studied using skinned muscle fibers or fragmented heavy SR (HSR) prepared from frog muscle, and compared with those on caffeine-induced one. Exposure of the skinned fibers to 5 microM Ag+ produced a rapid and large contraction in the presence of 0.043 mM free Mg2+. When Mg2+ concentration was increased to 0.86 mM, Ag+ led to a large transient contraction, combined with a small tonic one. The transient component was completely blocked by high Mg2+ (3.64 mM), but the tonic one was not. Ca2+-ATPase activity was not stimulated by increase of Mg2+ from 0.86 to 3.64 mM. Ag+ and caffeine induced a rapid Ca2+ efflux from HSR in a dose-dependent manner. RR over a range from 1 to 10 microM dose-dependently inhibited the Ca2+ efflux induced by 10 microM Ag+. Despite increase of RR to 30 microM, however, further inhibition of the Ca2+ efflux was not produced any more (77.8 +/- 12.2% inhibition). A 10 mM caffeine-induced efflux of Ca2+ was blocked slightly by only 0.5 microM RR and almost completely by 3 microM. A slight inhibition (about 28%) of the Ca2+-ATPase activity was observed in the presence of 10 microM Ag+ in 0.5 mg SR protein/ml of medium. RR and caffeine did not affect the enzyme activity. These results indicate that frog SR could induce a rapid release of Ca2+ upon Ag+ and caffeine, suggesting that Ag+ may have two different binding sites to release Ca2+; one is on Ca2+-induced Ca2+ release channel and the other on RR-insensitive site.