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M Shigekawa

Publications and source records attributed to M Shigekawa.

At least 73 records · Page 4Linked to original sources
PubMed1992

Phosphorylation of ryanodine receptors in rat myocytes during beta-adrenergic stimulation.

We studied beta-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [gamma-32P]ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the beta-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The beta-agonist-induced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during beta-adrenergic stimulation of cardiac myocytes.

Adrenergic beta-Agonists↗
PubMed1992

Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.

We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.

Animals↗
PubMed1992

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by 2,5-di(tert-butyl)-1,4-benzohydroquinone.

We characterized the interaction of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) with the sarcoplasmic reticulum (SR) Ca(2+)-ATPase from rabbit fast-twitch skeletal and canine cardiac muscles by examining the effect of this agent on the ATPase reaction. tBuBHQ at less than 10 microM inhibited ATP hydrolysis by both isoforms of Ca(2+)-ATPase by up to 80 and 90%, respectively. The half maximal inhibition of these enzymes was observed at about 1.5 microM tBuBHQ. Thus, this agent potently inhibits the fast-twitch skeletal and slow-twitch skeletal/cardiac isoforms of SR Ca(2+)-ATPase. tBuBHQ at 5-10 microM inhibited the rate of decomposition of the phosphoenzyme intermediate (EP), measured as a ratio between ATPase activity and the EP level in the steady state, by 35-40%. It also inhibited formation of EP by decreasing the rate of Ca2+ binding to the Ca(2+)-deficient, nonphosphorylated enzyme to about 1/8 of the control value. These results indicate that tBuBHQ has at least two sites of action in the reaction sequence for the SR Ca(2+)-ATPase.

Animals↗
PubMed1992

Altered agonist-induced Ca2+ mobilization in aortic smooth muscle cells from cardiomyopathic hamsters.

We compared the responses to the Ca(2+)-mobilizing effects of norepinephrine (NE), endothelin-1 (ET), and arginine vasopressin (AVP) of primary cultures of vascular smooth muscle cells (VSMCs) from aortas of young (30-40 days old) cardiomyopathic (Bio 14.6 strain) and age-matched control hamsters. In Bio 14.6 VSMCs, saturating concentrations of NE and ET elicited increases in the intracellular Ca2+ concentration ([Ca2+]i), which reached approximately 50 and 62% of those in control VSMCs. AVP elicited a significant increase in [Ca2+]i only in Bio 14.6 VSMCs. In contrast, 10 microM ionomycin induced similar Ca2+ responses in both types of cells in the absence of extracellular Ca2+. Ligand binding assay revealed that maximal binding values for alpha 1-adrenergic antagonists and ET in Bio 14.6 VSMCs were 59-64 and 85% of those in control VSMCs. Thus VSMCs from aortas of Bio 14.6 hamsters had reduced reactivity to NE and ET but increased response to AVP. The reduced Ca2+ response of Bio 14.6 VSMCs to NE appeared to be due mainly to a decrease in the receptor number.

Animals↗
PubMed1992

Cyclic AMP-dependent phosphorylation of the rat brain ryanodine receptor.

Phosphorylation of the rat brain ryanodine receptor was studied using a monoclonal antibody, Ry-1, against the cardiac ryanodine receptor. A large polypeptide with the same SDS-PAGE mobility as that of the canine cardiac receptor was detected in rat brain membranes by immunoblotting. The brain ryanodine receptor was solubilized from the microsomal membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and more than 85% of the solubilized receptor was immunoprecipitated by Ry-1. Immunoprecipitated receptors were phosphorylated by cAMP-dependent protein kinase. The ryanodine receptor was also expressed in cultured fetal rat brain neurons and was phosphorylated by treating the cells with dibutyryl cAMP. The number of cells showing a caffeine-induced Ca2+ transient was increased significantly in the phosphorylating condition. These results suggest that the Ca channel activity of the brain ryanodine receptor is regulated by cAMP-dependent phosphorylation.

Animals↗
PubMed1991

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.

Animals↗
PubMed1991

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.

Affinity Labels↗
PubMed1991

Activation of sarcoplasmic reticulum Ca(2+)-ATPase by Mn2+: a Mn2+ binding study.

We used Mn2+ as an analogue for Mg2+ to examine the minimum requirement of divalent cations for the rapid turnover of the sarcoplasmic reticulum ATPase. We measured the binding of Ca2+ and Mn2+ to the purified Ca(2+)-ATPase during steady-state hydrolysis of MnATP at 2 degrees C and pH 7. In the presence of 20 microM Ca2+, Mn2+ was as effective as Mg2+ in stimulating ATPase activity and the maximal activation of ATP hydrolysis was observed at 0.1 mM MnCl2. Under these conditions, 2 mol of Ca2+ were bound per mol of the ADP-sensitive phosphoenzyme, whereas no Ca2+ was bound to the ADP-insensitive phosphoenzyme. On the other hand, the stoichiometry for ATP-dependent binding of Mn2+ to these intermediates was about 1. We found that Mn2+ remained bound to the ADP-insensitive phosphoenzyme even in the presence of added chelator. In the absence of ATP, we detected a low level of Mn2+ binding, which reached 0.4 mol per mol of the phosphorylation site at 0.1 mM free Mn2+. We present evidence that this extra Mn2+ binding did not affect the rate of decomposition of the ADP-sensitive phosphoenzyme, which was the rate-limiting step for ATP hydrolysis under the conditions used.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Participation of H+ in the Ca2(+)-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase.

The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Protein-protein interaction of detergent-solubilized Ca2(+)-ATPase during ATP hydrolysis analyzed by low-angle laser light scattering photometry coupled with high-performance gel chromatography.

Protein-protein interaction of detergent-solubilized Ca2(+)-ATPase was examined, employing low-angle laser light scattering photometry coupled with high-performance gel chromatography. When solubilized with octa(ethylene glycol) mono-n-dodecyl ether (C12E8) and chromatographed in the presence of 0.3 mg/ml C12E8, the Ca2(+)-ATPase emerged as a single peak with an intermediate molecular weight between the monomer and the dimer, showing a dissociation-association equilibrium of the two components. In the presence of 50 micrograms/ml phosphatidylcholine and 0.3 mg/ml C12E8 at 0 degrees C, the Ca2(+)-ATPase (0.8 mg) emerged as the two distinct components with molecular weights of 125,000 +/- 2100 (n = 3) and 211 300 +/- 7300 (n = 3), indicating that there was no rapid interconversion between the monomer and the dimer. Under the latter conditions, addition of ATP induced fusion of two components. The apparent molecular weight of the fused peak shifted from the monomer to the dimer as the amount of protein increased. Addition of ADP or adenosine 5'-(beta, gamma-methylene triphosphate), however, did not induce such fusion of the peaks. The ATP-induced fusion of the peaks was not observed either in 5 mM CaCl2, the conditions in which the rate of ATP hydrolysis was extremely slow. Thus, the solubilized Ca2(+)-ATPase underwent a rapid interconversion between the monomer and the dimer during ATP hydrolysis. These results suggest that the protein-protein interaction during ATP hydrolysis is an intrinsic nature of Ca2(+)-ATPase and that such interaction may be important for Ca2+ transport by Ca2(+)-ATPase in the sarcoplasmic reticulum membranes.

Adenosine Triphosphate↗
PubMed1990

Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel.

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2(+)-dependent Cl- current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.

Amino Acid Sequence↗
PubMed1990

Mechanism for activation of the 4-nitrobenzo-2-oxa-1,3-diazole-labeled sarcoplasmic reticulum ATPase by Ca2+ and its modulation by nucleotides.

The mechanism for activation of sarcoplasmic reticulum ATPase by Ca2+ was investigated in 2 mM MgCl2 and 0.1 M KCl at pH 6.5 and 11 degrees C by using enzyme preparations in which a specific amino acid residue (Cys-344) was labeled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) [Wakabayashi, S., Imagawa, T., & Shigekawa, M. (1990) J. Biochem. (Tokyo) 107, 563-571]. We compared the kinetics of binding and dissociation of Ca2+ from the enzyme with those of the accompanying NBD fluorescence changes. The fluorescence rise following addition of Ca2+ proceeded monoexponentially. At 2-100 microM Ca2+ and in the absence of nucleotides, the Ca2(+)-induced fluorescence rise and Ca2+ binding to the enzyme proceeded at similar rates, which were almost independent of the Ca2+ concentration. In contrast, the fluorescence decrease induced by Ca2+ removal was slower than the Ca2+ dissociation, and both of these processes were inhibited markedly by increasing medium Ca2+. ATP by binding at 1 mol/mol of the phosphorylation site markedly accelerated both the Ca2(+)-induced fluorescence rise and Ca2+ binding, ADP and AMPPNP but not GTP also being effective. In contrast, ADP minimally affected the NBD fluorescence decrease and the Ca2+ dissociation. These data are consistent with a reaction model in which binding of Ca2+ occurs after the conformational transition of the free enzyme from a state (E2) having low affinity for Ca2+ to one (E1) having high affinity for Ca2+ and in which ATP bound at the catalytic site of E2, whose affinity for ATP is about 30-fold less than that of E1, accelerates this conformational transition.

Adenosine Triphosphatases↗
PubMed1990

Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition?

When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody.

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.

Animals↗
PubMed1990

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle.

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.

Adenosine Triphosphate↗