Masahiro Saitoh

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.

Protein kinase C negatively modulated by phorbol ester.

Pretreatment of protein kinase C with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and phospholipid resulted in complete inhibition of ATP/phosphotransferase activity, irreversibly. The inactivation by TPA required the phospholipid, and TPA alone did not cause inactivation. Ca2+ and diacylglycerol mimicked TPA. This action of TPA was not general for all protein kinases as it did not accelerate the inactivation of the catalytic subunit of cAMP‐dependent protein kinase by phospholipid. The addition of MgATP to the reaction mixture completely protected protein kinase C from being inactivated by TPA, in the presence of phospholipid. The nucleotide‐binding site of the enzyme was probably influenced by the binding of TPA and phospholipid.

Two phosphorylated forms of myosin in thrombin-stimulated platelets.

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.

Different sensitivities of Ca2+, calmodulin-dependent cyclic nucleotide phosphodiesterases from rabbit aorta and brain to dihydropyridine calcium channel blockers

The concentrations of the dihydropyridines, CD-349, nicardipine, and nimodipine, producing 50% inhibition of Ca2+, calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (CaPDE) from rabbit aorta in the absence of Ca2+-CaM complex were approximately 7 to 13-fold higher than these of aorta CaPDE in the presence of Ca2+-CaM complex and of the trypsin treated enzyme form. On the other hand, these dihydropyridine derivatives inhibited CaPDE from rabbit brain at much the same IC50 values seen in the absence and presence of the Ca2+-CaM complex and the trypsin-treated enzyme. Kinetic analysis revealed that these dihydropyridines inhibited the activities of CaPDEs from both the aorta and brain, competitively with cyclic GMP as substrate, and the Ki values of CD-349 for CaPDE from aorta or brain in the absence or presence of Ca2+-CaM complex and trypsin-treated enzyme were 9.6, 0.75, 0.75 or 0.69, 0.70, 0.66 microM, respectively. These results suggest that CaPDE from the rabbit aorta differs from this enzyme in the brain, with regard to the relationship between the dihydropyridine binding sites on CaPDE molecules and the domains regulated by the Ca2+-CaM complex or limited proteolysis.

Molecular Pharmacology of Myosin Light Chain Phosphorylation of Smooth Muscle and Nonmuscle Cells

Publisher Summary This chapter focuses on the molecular pharmacology of myosin light chain phosphorylation of smooth muscle and nonmuscle cells. Myosin light chain phosphorylation of smooth muscle and nonmuscle cells are catalyzed by both Ca, calmodulin dependent myosin light chain kinase, and protein kinase C. Purified smooth muscle myosin light chain kinase is inactivated by incubation with 5′-p-fluorosulfonylbenzyol adenosine, at 30°C. To clarify the relationship between the chemical structure and potency in inhibition of ML-9, and its derivatives, iodine-, bromine- and chlorine-substituted derivative at the naphthalene ring were examined for their inhibitory effect on myosin light chain kinase. Protein phosphorylation is an established major general mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli, but its effects are indirect and interrelationships among each protein phosphorylation system are complex. Diverse Ca2+-dependent cellular processes such as smooth muscle contraction and nonmuscle cell secretion are thought to be regulated by Ca2+ dependent protein phosphorylation systems. To investigate the quantitative aspects and molecular mechanisms of the calcium messenger system, the potent and specific inhibitors will be synthesized in studies on this cellular regulatory system.