Hiroyoshi Hidaka

1-(5-isoquinol inesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase c in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.

Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle unit cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.

Effects of Cilostazol, a Selective cAMP Phosphodiesterase Inhibitor on the Contraction of Vascular Smooth Muscle

The effects of cilostazol (OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quin olinone) on cyclic nucleotide metabolism and Ca2+-induced contraction of intact and skinned rabbit arterial smooth muscles were investigated. The concentrations of cilostazol producing 50% inhibition of cyclic adenosine monophosphate phosphodiesterase and Ca2+-dependent cyclic nucleotide phosphodiesterase were 0.4 microM and above 100 microM, respectively. This compound has no significant effect on adenylate cyclase in concentrations of up to 100 microM. Addition of cilostazol increased significantly the cAMP content without significant effect on cyclic guanosine monophosphate level of rabbit thoracic aorta in the presence of forskolin. Moreover, the ED50 value of cilostazol in relaxation of rabbit mesenteric arterial strips was decreased selectively by addition of 0.01 microM forskolin, which alone at this concentration has no effect on vascular contraction. Cilostazol of up to 30 microM did not suppress the Ca2+-induced contraction of the chemically skinned rabbit mesenteric artery. Therefore, cilostazol may produce the relaxation of intact vascular smooth muscle by its inhibition of cyclic adenosine monophosphate hydrolysis.

Effects of vinpocetine on cyclic nucleotide metabolism in vascular smooth muscle

A novel vasodilating agent, vinpocetine (14-ethoxycarbonyl-(3 alpha,16 alpha-ethyl)-14,15-eburnamenine) inhibits Ca2+-dependent phosphodiesterase, selectively, among the three forms of cyclic nucleotide phosphodiesterase identified in the rabbit aorta. The concentration of vinpocetine producing 50% inhibition of Ca2+-dependent phosphodiesterase activity was approximately 21 microM, both in the presence and absence of Ca2+-calmodulin (CaM). Increasing the concentration of CaM in the presence of Ca2+ did not prevent vinpocetine-induced inhibition of Ca2+-dependent phosphodiesterase, thereby indicating that vinpocetine inhibited the enzyme by interacting with the enzyme and not with CaM. To determine the influence of vinpocetine-induced inhibition of Ca2+-dependent phosphodiesterase on cyclic nucleotide metabolism in vascular smooth muscle, cyclic nucleotide levels in isolated rabbit aortic strips were also investigated. Addition of vinpocetine produced dose-dependent increases in only the cyclic GMP levels and there was no significant effects on the cyclic AMP levels. These results provide pharmacological evidence that Ca2+-dependent phosphodiesterase mainly hydrolyzes cyclic GMP in vascular smooth muscle. Vinpocetine may induce vascular relaxation by increasing cyclic GMP contents in vascular smooth muscle through selective inhibition of Ca2+-dependent phosphodiesterase.

Calmodulin and cell proliferation

Abstract The calmodulin content of synchronized Chinese hamster ovary (CHO-K 1 ) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G 1 phase, increased after the cells entered S phase and reached the maximum level at the late G 2 or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G 2 or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.

Distribution of S-100b protein in normal salivary glands and salivary gland tumors

Immunohistochemical studies were performed for the presence of S-100b protein in non-neoplastic and neoplastic salivary gland tissues by the peroxidase anti-peroxidase (PAP) method. Some cases of pleomorphic adenoma were investigated by immuno-electron microscopy. S-100b protein could not be detected in epithelial cells of intercalated ducts, acini, striated ducts and excretory ducts of non-neoplastic salivary gland. However, myoepithelial cells surrounding the acini and intercalated ducts were specifically stained by S-100b protein. In pleomorphic adenomas, S-100b protein-positive cells could be mostly observed in the myxoid and chondroid areas, and the basal layer cells of the double-layered ductal cells were also positive. In clear cell adenoma, the clear cells were also S-100b protein positive. In adenoid cystic carcinomas, S-100b protein-positive cells could be found in trabecular areas, but not in tumor cells showing cribriform-pattern. In other tumors (Warthins tumor, oxyphilic adenoma, basal cell adenoma, mucoepidermoid tumor and acinar cell carcinoma), S-100b protein positive cells were seldom observed. Immuno-electron microscopically, S-100b protein was diffusely distributed in the cytoplasm of myoepithelial cells as well as of tumor cells of pleomorphic adenoma, being distributed especially on the membrane of endoplasmic reticulum and the outer nuclear membrane.

Interaction of local anesthetics with calmodulin.

Abstract Dibucaine, tetracaine and lidocaine inhibited selectively Ca 2+ -calmodulin-induced activation of cyclic nucleotide phosphodiesterase with IC 50 values of 0.19, 0.44 and 4.80 mM, respectively and were less potent inhibitors in the absence of Ca 2+ . These drugs also inhibited other Ca 2+ -calmodulin-dependent enzyme such as myosin light chain kinase from chicken gizzard. [ 3 H]W-7 bound to purified calmodulin was displaced in a concentration-dependent manner by these compounds. Their 50% inhibitory concentrations were linearly related to their octanol-water partition coefficients, implying hydrophobic interaction between these drugs and calmodulin.

The modulatory role of myosin light chain phosphorylation in human platelet activation

Myosin 20 K-Da light chain phosphorylation in human platelets was found to be catalyzed by MLCK in the early phase during collagen activation. The effect of newly synthesized selective inhibitor of MLCK, ML-9, on collagen induced platelet activation was investigated. ML-9 delayed the time course of the myosin 20 K-Da light chain phosphorylation, sequentially led to a delay in aggregation, secretion and phosphorylation of the 40K-Da peptide, in a dose-dependent fashion. It is proposed that the MLCK catalyzed phosphorylation of myosin 20 K-Da light chain may be an initial response and if so may influence the sequent reactions in the activation of platelets with collagen.

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Abstract Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca 2+ -activated phospholipid-dependent protein kinase. Autoradiography showed that Ca 2+ -activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca 2+ -activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca 2+ -dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.

Ca2+-Activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.