Yamada K, Iwahashi K, Kase H
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Biochem Pharmacol. 1988 Mar 15;37(6):1161-6. doi: 10.1016/0006-2952(88)90525-4.
K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetr ahy dro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadi benzo[a,g]cycloocta[c,d,e]triden-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-alpha-acetyl-sn-glycero-phosphocholine (AGEPC)] induced protein phosphorylation, serotonin release and a rise in intracellular free calcium using washed rabbit platelets. In Ca2+-containing medium (1 mM CaCl2), AGEPC at 10(-10) and 10(-9) M markedly phosphorylated two proteins having molecular weights of 40,000 daltons (40 K protein) and 20,000 daltons (20 K protein) and evoked a marked rise in cytosolic free calcium. K-252a at 3 and 10 microM 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 Ca2+-free medium [1 mM ethylene glycol-bis(beta-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 microM 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-stimulated platelets, the inhibition of 20K protein phosphorylation in Ca2+-containing medium and of 40K protein phosphorylation in Ca2+-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.
K-252a，即(8R*,9S*,11S*)-(-)-9-羟基-9-甲氧基羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b,11a-三氮杂二苯并[a,g]环辛[c,d,e]三烯-1-酮，是一种从微生物中分离得到的吲哚咔唑化合物，能有效抑制部分纯化的酶和完整血小板中的蛋白激酶C。我们使用洗涤过的兔血小板，研究了该化合物对血小板活化因子[1-O-烷基-α-乙酰基-sn-甘油磷酸胆碱（AGEPC）]诱导的蛋白磷酸化、5-羟色胺释放以及细胞内游离钙升高的影响。在含Ca2+的培养基（1 mM CaCl2）中，10(-10)和10(-9) M的AGEPC能显著磷酸化两种分子量分别为40,000道尔顿（40K蛋白）和20,000道尔顿（20K蛋白）的蛋白，并引起胞质游离钙显著升高。3和10 microM的K-252a对20K蛋白磷酸化产生浓度依赖性抑制，但对40K蛋白磷酸化仅产生轻微抑制。K-252a抑制了未刺激血小板中20K蛋白的基础磷酸化，且对AGEPC引起的细胞内游离钙升高无显著影响。据此证据可以认为，K-252a可能直接作用于肌球蛋白轻链激酶，从而抑制2-0K蛋白磷酸化。在无Ca2+的培养基[1 mM乙二醇双（β-氨基乙醚）-N,N,N',N'-四乙酸（EGTA）]中，10(-8) M的AGEPC主要磷酸化40K蛋白，尽管20K蛋白的磷酸化和胞质游离钙略有增加。1-10 microM的K-252a对40K蛋白磷酸化产生浓度依赖性抑制。这些结果表明，K-252a在兔血小板中既是蛋白激酶C的抑制剂，也是肌球蛋白轻链激酶的抑制剂。在AGEPC刺激的血小板中，在含Ca2+培养基中对20K蛋白磷酸化的抑制以及在无Ca2+培养基中对40K蛋白磷酸化的抑制与K-252a对5-羟色胺释放的抑制密切相关。这些结果强烈表明，这两种蛋白的磷酸化可能是AGEPC刺激的血小板中5-羟色胺释放的先决条件。
