Biomedical subjects

H Takisawa

Publications and source records attributed to H Takisawa.

At least 19 recordsLinked to original sources

PubMed1992

Interaction of the low-molecular-mass, guanine-nucleotide-binding protein with the actin-binding protein and its modulation by the cAMP-dependent protein kinase in bovine platelets.

Platelets have been shown to possess several, different, low-molecular-mass, guanine-nucleotide-binding proteins (G-proteins) with molecular masses about 20-30 kDa. We report here that a 25-kDa G-protein copurified with the bovine platelet actin-binding protein (ABP), a cross-linker of actin filaments which is known to generate the three-dimensional network of actin. Both the G-protein and ABP were recovered in a fraction that was insoluble in Triton X-100 and were extracted in 0.6 M NaCl. Gel-filtration chromatography of the high-salt extract and rechromatography in a low-salt solution indicated that the two proteins may be associated with each other. The association of the two proteins was suggested by cosedimentation of the G-protein with the actin gel formed by actin and ABP. The amounts of the cosedimented G-protein and ABP was unaffected by guanosine-5'-O-[beta-thio]diphosphate and guanosine-5'-O-[gamma-thio]triphosphate, but the G-protein, not ABP, was partially released from the actin gel by phosphorylating ABP with cAMP-dependent protein kinase. Thus, the association of the two proteins was affected by modification of ABP, but not by modification of G-proteins. The physiological significance of the possible association of the two proteins might be that the membrane skeleton functions as a modulator of the G-protein, rather than that the G-protein modulates the function of the membrane skeleton which comprises ABP.

Affinity Labels↗

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

Contractile activity and fluorescence changes in fluo-3-loaded isolated ventricular myocytes.

Isolated rat ventricular myocytes were loaded with fluo-3 which did not result in a loss of beating activity, in order to record the changes in the fluorescence and contractile parameters. Changes in the beating activity induced by various reagents in perfusing medium were related to variations in the peak intensity of fluorescence time course and possibly to changes in myofibrillar ATPase activity. Isoproterenol stimulated, whereas 2,3-butanedione monoxime (BDM) and a medium with a low Ca2+ concentration suppressed the contractile activity by increasing and reducing the Ca2+ transient, respectively, and, in the presence of BDM, a decrease in the maximum ATPase activity also contributed the suppressive effect.

Aniline Compounds↗

PubMed1990

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation.

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.

Animals↗

PubMed1990

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle.

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.

Animals↗

PubMed1990

Repression of serotonin secretion by an endogenous Ca2(+)-activated protease in electropermeabilized bovine platelets.

Micromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca2(+)-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35 degrees C in 100 microM Ca2+ without Mg-ATP. The half-maximal effect was observed with about 45 microM Ca2+ in the preincubation medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca2+. Electron microscopic observation revealed that disorganization of the cytoskeletal structures, in particular of the membrane undercoat and the network of microfilaments, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca2(+)-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100 microM Ca2+ but not in the presence of Ca2+ and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantitatively correlated with the degree of impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a 260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 110-kDa components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Bi-directional regulation of dephosphorylation of cAMP-dependent phosphorylated proteins by cAMP and calcium in permeabilized rat heart cells.

We studied the regulation of dephosphorylation of cAMP-dependent phosphorylated proteins of isolated, permeabilized (skinned) myocardial cells from adult rat. Staurosporine, a potent inhibitor of protein kinase, inhibited cAMP-dependent phosphorylation of phospholamban and troponin-I, the key proteins in the control of contraction and relaxation of the myocardial cells. Staurosporine antagonized the stimulatory action of cAMP on the spontaneous beating of the myocytes accompanied by dephosphorylation of phospholamban but not of troponin-I at pCa 7-8. In cold ATP dilution experiments with apparent stoppage of protein phosphorylation, dephosphorylation of phospholamban was accelerated both by Ca2+ and staurosporine but that of troponin-I took place only in the presence of Ca2+ ion (pCa less than 6.5). These phenomena suggest a bi-directional regulation of dephosphorylation of the key proteins by the intracellular messengers cAMP and Ca2+.

Adenosine Triphosphate↗

PubMed1988

Occluded Ca2+.

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Adenosine Triphosphate↗

PubMed1987

Beta-adrenergic regulation of contractility and protein phosphorylation in spontaneously beating isolated rat myocardial cells.

Spontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between beta-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by beta-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when beta-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the beta-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were membrane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phosphorylation of these five proteins was stimulated by isoproterenol. The effect of isoproterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of the stimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional beta-adrenergic receptor and cellular responses to beta-adrenergic stimulation. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.

Adenosine Triphosphate↗

PubMed1987

A possible role of protein phosphorylation in the inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The Ca2+-induced Ca2+ release channel in the heavy fraction of the sarcoplasmic reticulum (SR) from rabbit skeletal muscle is inactivated during ATP-dependent Ca2+ uptake (Morii, H., Takisawa, H., & Yamamoto, T. (1985) J. Biol. Chem. 260, 11536-11541). AMP, one of the adenine nucleotides which activate the Ca2+ release, delayed the onset of the channel inactivation when added early during the course of the Ca2+ uptake. However, AMP could no longer activate the channel but accelerated the inactivation when added during the later phase of the Ca2+ uptake. In SR passively loaded with Ca2+, the Ca2+ channel which had been activated by AMP and Ca2+ was not spontaneously inactivated. Similarly, during GTP-dependent Ca2+ uptake, the channel activated by AMP was not inactivated. In addition acid phosphatase markedly delayed the onset of the inactivation during ATP-dependent Ca2+ uptake, without affecting Ca2+ ATPase activity or GTP-dependent Ca2+ uptake by heavy SR. The effect of the phosphatase was completely blocked by ruthenium red, a potent inhibitor of the channel. These results suggest that the channel is inactivated through an ATP-dependent process, presumably phosphorylation of proteins in the SR membrane. This was supported by the findings that the reactivation of the inactivated channel by added Ca2+ was markedly accelerated by the addition of acid phosphatase and that several proteins of heavy SR were phosphorylated during ATP-dependent Ca2+ uptake.

Acid Phosphatase↗

PubMed1986

Distinct occurrence of phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release and inositol 1,4,5-triphosphate-induced release in ATP-dependent Ca2+-transporting platelet microsomes.

The effects of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and inositol 1,4,5-triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca2+-transporting microsomes prepared from ox platelets were investigated. Under optimal conditions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by InsP3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP3 appeared to act as a Ca2+ ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ca2+ from 0.1 microM to 10 microM and it was completely inhibited by 10 microM Ca2+. PtdInsP2-induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthenium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+ had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+ from platelet microsomes more effectively than InsP3 by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.

Animals↗

PubMed1985

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport.

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.

Adenine Nucleotides↗

PubMed1985

Inhibition of hydrolysis of phosphorylated Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum by Ca2+ inside and outside the vesicles.

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.

Adenosine Triphosphate↗

PubMed1979