Kageyama M, Mori T, Yanagisawa T, Taira N
Department of Pharmacology, Tohoku University School of Medicine, Sendai, Japan.
J Pharmacol Exp Ther. 1991 Dec;259(3):1019-26.
We assessed the specificity of staurosporine, a putative protein kinase C inhibitor, for the enzyme in intact porcine coronary arteries by examining its effects on changes in intracellular free calcium level ([Ca++]i) and force induced by a phorbol ester, high KCl and caffeine. [Ca++]i was measured simultaneously with force by the fura-2 microfluorimetric method. Phorbol 12,13-dibutyrate (PDBu, 10(-6) M) produced a slowly developing and sustained contraction; however, [Ca++]i only increased slightly and transiently in the initial phase and then decreased gradually. On the other hand, 90 mM KCl elicited a contraction with a sustained increase in [Ca++]i. Staurosporine (10(-10) to 10(-7) M) applied 20 min before the addition of PDBu inhibited the PDBu-induced changes in [Ca++]i and force in a concentration-dependent manner. In contrast, staurosporine only at 10(-7) M reduced an increase in [Ca++]i and contractile force produced by 90 mM KCl. The inhibitory effects of staurosporine on PDBu- and KCl-induced contractions depended on exposure time. The [Ca++]i-force relationship obtained with variable concentrations of KCl was shifted slightly to the right by staurosporine at 10(-7) M. Furthermore, staurosporine even at 10(-7) M did not affect an increase in [Ca++]i by caffeine (25 mM), although it slightly attenuated a caffeine-induced contraction. These results suggest that staurosporine is a relatively specific inhibitor of protein kinase C in intact arteries at lower concentrations. At higher concentrations it may have actions unrelated to its inhibitory effect of protein kinase C, which include the inhibition of calcium influx into smooth muscle cells through the voltage-dependent Ca++ channel.
我们通过研究星形孢菌素（一种假定的蛋白激酶C抑制剂）对完整猪冠状动脉中该酶的特异性，检测了其对佛波酯、高钾和咖啡因诱导的细胞内游离钙水平（[Ca++]i）变化及张力的影响。采用fura-2显微荧光法同时测量[Ca++]i和张力。佛波醇12,13-二丁酸酯（PDBu，10(-6) M）引起缓慢发展且持续的收缩；然而，[Ca++]i仅在初始阶段轻微且短暂增加，随后逐渐下降。另一方面，90 mM KCl引起收缩并伴有[Ca++]i持续增加。在加入PDBu前20分钟应用星形孢菌素（10(-10)至10(-7) M），以浓度依赖方式抑制PDBu诱导的[Ca++]i和张力变化。相比之下，仅10(-7) M的星形孢菌素可降低90 mM KCl引起的[Ca++]i增加和收缩力。星形孢菌素对PDBu和KCl诱导收缩的抑制作用取决于暴露时间。10(-7) M的星形孢菌素使不同浓度KCl获得的[Ca++]i-张力关系曲线略有右移。此外，即使10(-7) M的星形孢菌素也不影响咖啡因（25 mM）引起的[Ca++]i增加，尽管它略微减弱了咖啡因诱导的收缩。这些结果表明，在较低浓度下，星形孢菌素是完整动脉中蛋白激酶C的相对特异性抑制剂。在较高浓度下，它可能具有与其对蛋白激酶C的抑制作用无关的作用，包括抑制通过电压依赖性Ca++通道的钙流入平滑肌细胞。
