Kase Hiroshi

Parallel inhibition of platelet-activating factor-induced protein phosphorylation and serotonin release by K-252a, a new inhibitor of protein kinases, in rabbit platelets

Abstract K-252a, (8 R ,9 S ,11 S )-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H ,8 H ,11 H -2,7b,11a-triazadi benzo[ a , g ]cycloocta[ c , d , e ]trinden-1-one, an indole carbazol compound isolated from microbial origin, potently inhibits protein kinase C in partially purified enzyme and intact platelets. We examined the effects of this compound on platelet-activating factor [1- O -alkyl- α -acetyl-sn-glycero-phosphocholine (AGEPC)] induced protein phosphorylation, serotonin release and a rise in intracellular free calcium using washed rabbit platelets. In Ca 2+ -containing medium (1 mM CaCl 2 ), AGEPC at 10 −10 and 10 −9 M markedly phosphorylated two proteins having molecular weights of 40,000 dallons (40 K protein) and 20,000 dallons (20 K protein) and evoked a marked rise in cytosolic free calcium. K-252a at 3 and 10 μM caused a concentration-dependent inhibition in the 20 K protein phosphorylation but caused only slight inhibition in the 40 K protein phosphorylation. K-252a inhibited the basal phosphorylation of 20 K protein obtained in non-stimulated platelets, and caused no significant alteration in the rise of intracellular free calcium evoked by AGEPC. It can be considered, from this evidence, that K-252a may act directly on myosin light chain kinase, resulting in the inhibition of 20 K protein phosphorylation. In Ca 2+ -free medium [1 mM ethylene glycol-bis( β -aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA)], AGEPC at 10 −8 M predominantly phosphorylated 40K protein, although phosphorylation of 20K protein and cytosolic free calcium were increased slightly. K-252a at 1–10 μM caused a concentration-dependent inhibition in the 40K protein phosphorylation. These results indicate that K-252a functions as an inhibitor of both protein kinase C and myosin light chain kinase in rabbit platelets. In AGEPC-stimuIated platelets, the inhibition of 20K protein phosphorylation in Ca 2+ -containing medium and of 40K protein phosphorylation in Ca 2+ -free medium was closely correlated with the inhibition of serotonin release by K-252a. These results strongly suggest that the phosphorylation of these two proteins may be a prerequisite for serotonin release in AGEPC-stimulated platelets.

Inhibition by new anthraquinone compounds, K-259-2 and KS-619-1, of calmodulin-dependent cyclic nucleotide phosphodiesterase

Abstract K-259-2 and KS-619-1, novel anionic anthraquinone metabolites isolated from culture broth of microorganisms, inhibited activation of bovine brain phosphodiesterase induced by calmodulin (CaM), sodium oleate, or limited proteolysis with almost equal potency. The inhibition of calmodulinactivated phosphodiesterase (CaM-PDE) by K-259-2 or KS-619-1 was overcome by a higher concentration of CaM. Direct interaction of K-259-2 and KS-619-1 with CaM was confirmed through use of hydrophobic fluorescent probes. Kinetic analysis revealed that the inhibition of the trypsin-activated phosphodiesterase was competitively inhibited by K-259-2 or KS-619-1 with respect to cAMP. Addition of a lower amount of either phosphatidylserine or sodium oleate to the reaction mixture was efficacious in attenuating the inhibition of the CaM-PDE by W-7, chlorpromazine, trifluoperazine, compound 48 80 , or R-24571 but, in contrast, had little or no effect on the inhibition by K-259-2 or KS-619-1. In conclusion, K-259-2 and KS-619-1, unlike so-called CaM antagonists, do not interact with phosphatidylserine or sodium oleate and it appears that these novel anthraquinone compounds inhibit the enzyme not only via CaM antagonism but possibly also by interacting directly with the enzyme.