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.