Biomedical subjects

M Shigekawa

Publications and source records attributed to M Shigekawa.

At least 55 records · Page 3Linked to original sources

PubMed1996

Dystrophin-glycoprotein complex purified from hamster cardiac muscle. Comparison of the complexes from cardiac and skeletal muscles of hamster and rabbit.

The dystrophin-glycoprotein complex was isolated from hamster ventricular muscle by a method involving homogenization of muscle directly in the presence of 1% digitonin, followed by chromatography on succinylated wheat germ agglutinin agarose, Diethyl aminoethyl (DEAE) cellulose, and/or immunoaffinity agarose. Protein yield of the DEAE cellulose-purified dystrophin-glycoprotein complex was 120 +/- 30 (n = 3) micrograms per 5 g hamster ventricular muscle. The cardiac dystrophin-glycoprotein complex, unlike the skeletal muscle counterpart, could not be solubilized from a microsomal fraction with digitonin or some other detergents. By sodium dodecyl sulfate gel electrophoresis, protein composition of the dystrophin-glycoprotein complexes from hamster cardiac muscle was found to be significantly different from that of rabbit skeletal muscle which has been extensively studied. This difference mainly arises from the species difference, because in hamster the cardiac and skeletal muscle complexes exhibited essentially the same protein composition. In rabbit, on the other hand, there are differences between the cardiac and skeletal complexes in the relative abundance of 60 and 64 kDa proteins and in the apparent M(r) of alpha-dystroglycan. We found that the content of the dystrophin-glycoprotein complex, estimated by quantitative immunoblot assay, is at least 5 times more abundant in cardiac than in skeletal muscle in hamster and rabbit.

Animals↗

PubMed1995

Cytoplasmic domain of the ubiquitous Na+/H+ exchanger NHE1 can confer Ca2+ responsiveness to the apical isoform NHE3.

The Na+/H+ exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca(2+)-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H+ modifier site due to direct binding of Ca2+/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e. NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3 (N1N3), and NHE3 with the cytoplasmic domain of NHE1 (N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (< 80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Among stimuli tested (ionomycin, alpha-thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca2+ mobilization and early activation of 22Na+ uptake, implying that Ca2+ is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of 22Na+ uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pHi sensitivity (approximately 0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHi sensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca(2+)-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H(+)-modifier site of a different isoform, NHE3.

Amiloride↗

PubMed1995

Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger.

Although the Na+/Ca2+ exchanger is one of the major Ca2+ extrusion systems in excitable tissues, little is known about its regulation via protein phosphorylation. We now present evidence that the Na+/Ca2+ exchanger is phosphorylated in quiescent and growth factor-stimulated cultured aortic smooth muscle cells. The Na+/Ca2+ exchanger was isolated from 32P-labeled cells by immunoprecipitation with a specific polyclonal antibody. Phosphorylation of the exchanger was increased by up to 1.7-fold in response to platelet-derived growth factor-BB (PDGF-BB), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA). However, angiotensin II did not enhance the phosphorylation significantly. The extent of phosphorylation appeared to correlate with the growth factor-induced increase in cell 1,2-diacylglycerol. At least four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide map analysis of the phosphorylated exchanger, suggesting that phosphorylation occurred at multiple sites. PDGF-BB and PMA increased phosphorylation of the same phosphopeptides (in particular P1). Phosphorylated amino acids were exclusively serine residues in both quiescent and stimulated cells. We found that growth factors enhanced Na+/Ca2+ exchange activity and that there was a good correlation between the growth factor-induced stimulations of phosphorylation and exchange activity. PDGF-BB-induced activation of the exchanger was abolished by prior long treatment of cells with PMA. These results suggest that the Na+/Ca2+ exchanger is activated by protein kinase C-dependent phosphorylation in response to growth factors in vascular smooth muscle cells.

Animals↗

PubMed1995

Mechanisms of reoxygenation-induced calcium overload in cardiac myocytes: dependence on pHi.

This study investigated the selective effects of intracellular (pHi) or extracellular change in pH on reoxygenation-induced Ca2+ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICR mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was "reoxygenated" and the dynamics in intracellular Ca2+ concentration ([Ca2+]i) and pHi were monitored using the fluorescent dyes fura-2 and 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pHi progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pHi and a marked increase in [Ca2+]i (1884 +/- 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pHi recovery and significantly suppressed reoxygenation-induced Ca2+ overload (1170 +/- 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+]i was not reduced significantly compared with the control (1790 +/- 122 nM, P = 0.130). In addition, inhibition of Na(+)-H+ exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca2+]i (985 +/- 102 nM, P = 0.004). These results suggest that reoxygenation-induced Ca2+ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes.

Acids↗

PubMed1995

Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

Animals↗

PubMed1994

Ca(2+)-ATPase distributes differently in cardiac sarcolemma than dihydropyridine receptor alpha 1 subunit and Na+/Ca2+ exchanger.

We have investigated the distribution of the sarcolemmal Ca2+ transporters in hamster and dog ventricular myocytes by immunocytochemical and membrane fractionation techniques. The data suggest that the DHP receptor alpha 1 subunit and the Na+/Ca2+ exchanger are present in surface sarcolemma as well as T-tubule membranes located at the cardiac dyads. Compared with these Ca2+ transporters, the sarcolemmal Ca(2+)-ATPase is much less abundant in the latter fraction. Thus the sarcolemmal Ca(2+)-ATPase seems to be located predominantly in surface sarcolemma.

Animals↗

PubMed1994

Multiple mechanisms of arachidonic acid release in Chinese hamster ovary cells transfected with cDNA of substance P receptor.

We investigated the release of [3H]arachidonic acid ([3H]AA) and its relationship to the formation of [3H]inositol trisphosphate ([3H]IP3) elicited by substance P (SP) in prelabeled Chinese hamster ovary cells stably expressing the SP receptor. Activation of the SP receptor resulted in a concentration- and time-dependent stimulation of [3H]AA release. Half-maximal release was obtained at 10(-9) M, comparable to that for [3H]IP3 formation reported previously, and the maximal release effected by 0.1 microM SP was 8 to 10-fold above the basal value. Both the [3H]AA release and the [3H]-IP3 accumulation stimulated in the cells by 0.1 microM SP were concentration-dependently blocked with the specific SP receptor antagonist CP-96,345, with IC50 values of 2.5 and 0.4 microM, respectively. The time course of [3H]AA release showed a biphasic pattern: an initial rapid release essentially independent of Ca2+, followed by a sustained release markedly suppressed by removal of extracellular Ca2+ or chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA). While pretreatment with pertussis toxin (200 ng/mL, 6 hr) did not block [3H]IP3 formation, it did reduce [3H]AA release by 50% at 1 and 10 min after SP stimulation. Treatment of the cells with a phorbol ester, a protein kinase C activator, augmented the SP-stimulated [H]AA release, and sphingosine, a protein kinase C inhibitor, reversed the phorbol ester-potentiated [3H]AA release, but not the release stimulated by SP alone, suggesting a synergistic effect of protein kinase C on SP-stimulated AA release. These results demonstrate that SP, acting at the SP receptor, stimulates [3H]AA release via mechanisms that are (1) mediated by a pertussis toxin-sensitive G-protein, (2) dependent on extracellular Ca2+, and (3) enhanced by activation of protein kinase C.

Animals↗

PubMed1994

The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites.

The Na+/H+ exchange activity (NHE1 human isoform) is rapidly activated in response to growth factors and hyperosmotic stress. To get insight into the mechanism of NHE1 activation, we studied the direct interaction of a ubiquitous Ca(2+)-dependent regulatory factor, calmodulin (CaM) with NHE1. Binding experiments with CaM-Sepharose, as well as fluorescence measurements with dansylated CaM, revealed that the NHE1 cytoplasmic domain strongly binds CaM in a Ca(2+)-dependent manner. Fusion protein analysis with deletion mutants provided evidence for high (Kd approximately 20 nM) and intermediate (Kd approximately 350 nm) affinity CaM-binding sites located in neighboring regions of NHE1 (amino acids 636-656 and 657-700). To assess a regulatory role of CaM-binding sites, several cDNAs having deletion and point mutations in the high affinity site were generated and expressed in the exchanger-deficient fibroblast cell line PS120. Deletion and point mutations of positively charged residues of the high affinity CaM-binding site resulted in up to 50 and 80% reductions of cytoplasmic alkalinization caused by growth factors (alpha-thrombin, etc.) and 100 mM sucrose, respectively. In these mutants, the reduction in alkalinization was apparently in proportion to that of the CaM-binding ability. These results suggest that binding of Ca2+/CaM to the high affinity site is involved at least partly in the activation of NHE1 in response to different extracellular signals.

Amino Acid Sequence↗

PubMed1994

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective.

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.

Binding Sites↗

PubMed1994

Growth factor activation and "H(+)-sensing" of the Na+/H+ exchanger isoform 1 (NHE1). Evidence for an additional mechanism not requiring direct phosphorylation.

Growth factors stimulate the Na+/H+ exchange activity (NHE1 human isoform) and at the same time increase the phosphorylation state of the exchanger at serine residues. To determine the role of NHE1 phosphorylation, a set of deletion and point mutants has been generated. Functional characterization of deletion mutants expressed in fibroblastic cells revealed that the cytoplasmic region between amino acids 567 and 635 is required for both growth factor-induced cytoplasmic alkalinization and maintenance of high intracellular pH (pHi) sensitivity. In contrast to the loss of growth factor activation and pHi sensitivity caused by the deletion of amino acids 567-635, the same deletion had no apparent effect on the pattern of growth factor-induced phosphorylation. In addition, individual replacement of any of the serine residues between amino acids 567 and 635 with alanine also had no effect on growth factor activation of the exchange activity. Comparison of phosphopeptide maps for the wild type with those for the internal deletion mutant exchangers and the expressed cytoplasmic domain revealed that all major in vivo phosphorylation sites including growth factor-sensitive ones map to the cytoplasmic tail (amino acids 636-815). Deletion of these sites preserves high pHi sensitivity and reduces by only 50% growth factor-induced cytoplasmic alkalinization. Taken together, these data support the existence of a mechanism not requiring direct phosphorylation of NHE1, by which growth factor signals transmit to the "H(+)-sensor" and control the set point value of the exchanger. We propose that a regulatory factor(s) controls NHE1, presumably through its interaction with the critical cytoplasmic region prior to amino acid 635.

Cells, Cultured↗

PubMed1994

Regulation of Ca2+ entry in rat aortic smooth muscle cells in primary culture.

We characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) maintained in primary culture by measuring uptake of 45Ca2+ or Mn2+ from a normal balanced salt solution and the extracellular Ca(2+)-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) in a medium [high pH (pH 8.8)/high Mg2+ (20 mM) medium containing a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin] that inhibits removal of Ca2+ from the cytoplasm. Such measurements in the presence or absence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive voltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under resting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in these SMCs resulted in activation of neither DHP-sensitive nor -insensitive Ca2+ entry, but rather resulted in marked suppression of the former. Utilizing the DHP-sensitive extracellular Ca(2+)-induced increase in [Ca2+]i as a monitor of activity of the DHP-sensitive VGCCs, we investigated the effects of protein kinase activators and phosphatase inhibitors on the regulation of these VGCCs. We found that the DHP-sensitive VGCCs were inhibited by endothelin-1 through the activation of protein kinase C. We also found that they were inhibited by 8Br-cGMP, okadaic acid, and calyculin A.

Animals↗

PubMed1993

Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart.

In ventricular muscle from 30- to 60-day-old Bio 14.6 cardiomyopathic hamsters, dystrophin-associated glycoproteins of 43, 50 and 150 kDa are markedly reduced in abundance. In particular, the 50-kDa glycoprotein is totally deficient in the sarcolemma of myopathic ventricular myocytes as revealed by immunofluorescence microscopy. The dystrophin-glycoprotein complex formation is defective in the cardiomyopathic hamster heart, because dystrophin and the glycoproteins behave independently when digitonin-solubilized ventricular homogenates are fractionated on wheat germ agglutinin beads or anti-dystrophin immunoaffinity beads.

Animals↗

PubMed1993

Altered membrane-dystrophin association in the cardiomyopathic hamster heart muscle.

In the ventricular muscle from 30- to 40-day-old Bio 14.6 cardiomyopathic hamsters, the concentration of dystrophin was about 50% less than the normal controls. Despite its reduced content, extraction of dystrophin from the myopathic muscle into the microsomal fraction was threefold higher as compared to the control. These results suggest that abnormality exists in the association of dystrophin with sarcolemmal membranes in the myopathic muscle. No obvious difference, however, was observed between normal and myopathic myocytes in their immunofluorescence staining patterns.

Animals↗

PubMed1993

Maxi K+ channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells.

By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca(2+)-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274 +/- 4 pS in symmetrical 140 mM KCl at 24-26 degrees C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 microM Ca2+, 50 microM cGMP and 1 mM magnesium adenosine triphosphate. At -50 mV and 1 microM Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. alpha-Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery.

Adenosine Triphosphate↗

PubMed1993

Cloning of the rat aortic smooth muscle Na+/Ca2+ exchanger and tissue-specific expression of isoforms.

The amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100,676 and 103,200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.

Alternative Splicing↗

PubMed1993

Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart.

We determined the contents of L-type calcium channels (LCC) and other membrane proteins in ventricular homogenates and microsomes prepared from hearts of 30- to 70-day-old Syrian cardiomyopathic (Bio 14.6) and normal hamsters. Quantitative immunoblot assay revealed that myopathic microsomes, as compared with normal controls, were enriched about twofold with the alpha 1-subunit of LCC, the ryanodine receptor calsequestrin, and Na(+)-K(+)-adenosinetriphosphatase (ATPase), whereas the contents of these proteins in ventricular homogenates were not different. In contrast, Na(+)-H+ antiporter and sarcoplasmic reticulum (SR) Ca(2+)-ATPase showed no difference in their contents in both homogenates and microsomes. Radioligand binding assay further showed no significant difference in the number of binding sites for [3H]prazosin, [125I]iodocyanopindolol, and [3H]saxitoxin between myopathic and normal microsomes. These result suggest that whereas membrane densities of LCC and the other proteins examined are not increased in myopathic cardiomyocytes, T-tubule/junctional SR membranes are more easily extracted from them by mechanical disruption. This, together with 1.5-fold higher yield of microsomal fractions from myopathic heart muscle, shows that abnormality exists in the mechanical property of cell membrane in the myopathic heart.

Animals↗

PubMed1993

[Physiological functions of endoplasmic and sarcoplasmic reticulum Ca pump and pharmacology of inhibitors of the pump].

This review is derived from the symposium held at the 66th Annual Meeting of the Japanese Pharmacological Society (March, 1993). The symposium consisted of six invited papers whose general theme was the application of recently found ATPase inhibitors selective to SR- and ER-Ca(2+)-ATPase to the analyses of the physiological and pharmacological roles of endoplasmic and sarcoplasmic reticulum Ca stores. Inhibitors used were: thapsigargin, cyclopiazonic acid, 2,5-di-(t-butyl)-1,4-benzohydroquinone and 3',3",5',5"-tetraiodosulfophthalein. Gingerol was found to facilitate the action of the ATPase. In either smooth, cardiac or skeletal muscle, sympathetic neurons or several cell lines these inhibitors affected a variety of cell functions and conditions such as contraction, ionic conductance and excitability of the plasma membrane, regulation of intracellular free Ca2+ concentration, transport of viral glycoprotein to the cell surface. Many of these studies utilized either single or cultured cell preparations or skinned muscle. These inhibitors were shown to be useful tools for investigating the SR and ER functioning as Ca sources or Ca sequestrating pumps, and further for estimating the contribution of ER or SR to regulating the flux of Ca2+ and other ions through the plasma membrane. Results of analyses using these inhibitors are discussed.

Animals↗

PubMed1993

[Structure and function of cardiac sarcoplasmic reticulum Ca(2+)-ATPase].

The sarcoplasmic reticulum (SR) calcium pump plays a major role in the beat-to-beat removal of cytoplasmic Ca2+ in mammalian cardiac myocytes. The cardiac SR Ca(2+)-ATPase is one of the isoforms of the endoplasmic reticulum (ER) Ca(2+)-ATPases that are encoded by a gene family, consisting of at least three separate genes (SERCA1, SERCA2 and SERCA3). The high similarity in primary structure of these isoforms suggests that the basic structure-function relationship in these isoforms is also similar, although some differences have been noted between the functions of these isoforms. These isoforms are expressed in a tissue selective manner and change their expression during ontogeny or in response to physiological and pathological stimuli. In this article, we review the recent advances in the study of the cardiac SR Ca(2+)-ATPase in terms of its structure and function, and its expression.

Animals↗