Pucéat M, Hilal-Dandan R, Strulovici B, Brunton L L, Brown J H
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636.
J Biol Chem. 1994 Jun 17;269(24):16938-44.
We have immunologically identified the isoforms of protein kinase C (PKC) present in neonatal and adult rat cardiomyocytes and examined their regulation by hormones and phorbol ester. Both cell types express the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent epsilon- and delta-PKC isoforms. The atypical zeta-PKC isoform is also expressed in neonatal, but only weakly in adult cells. Stimulation of the alpha 1-adrenergic or purinergic receptor with phenylephrine or ATP, respectively, increases membrane-associated immunoreactivity of both epsilon- and delta-PKC in neonatal and adult cells; endothelin and carbachol are also effective in adult cells. In contrast, none of the agonists leads to increases in membrane-associated alpha-PKC in cardiomyocytes. PKC zeta is also unaffected by receptor stimulation. The phorbol ester phorbol 12-myristate 13-acetate causes redistribution and subsequently down-regulation of alpha-, epsilon-, and delta- but not zeta-PKC. The three isoforms are down-regulated at distinctively different rates, with alpha-PKC being the most rapid and epsilon-PKC the slowest. We used selective down-regulation of alpha-, epsilon-, and delta-PKC to investigate the role of these isoforms in PKC phosphorylation-dependent events in neonatal myocytes. Our findings suggest that epsilon-PKC is responsible for the phenylephrine-induced phosphorylation of MARCKS, an endogenous PKC-specific substrate. In contrast, agonist-induced c-fos expression is unlikely to be mediated by epsilon-PKC since the response is rapidly down-regulated and apparently Ca(2+)-dependent. Our finding that the PKC isoforms are differentially responsive to neurohormones suggests that they play distinct and specific roles in cardiac function.
我们已通过免疫学方法鉴定了新生大鼠和成年大鼠心肌细胞中存在的蛋白激酶C（PKC）同工型，并研究了激素和佛波酯对它们的调节作用。两种细胞类型均表达钙依赖性α-PKC以及钙非依赖性ε-PKC和δ-PKC同工型。非典型ζ-PKC同工型在新生细胞中也有表达，但在成年细胞中表达较弱。分别用去氧肾上腺素或ATP刺激α1-肾上腺素能受体或嘌呤能受体，可增加新生细胞和成年细胞中ε-PKC和δ-PKC的膜相关免疫反应性；内皮素和卡巴胆碱对成年细胞也有效。相比之下，这些激动剂均不会导致心肌细胞中膜相关α-PKC增加。PKCζ也不受受体刺激的影响。佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯会导致α-PKC、ε-PKC和δ-PKC重新分布，随后使其下调，但ζ-PKC不受影响。这三种同工型以明显不同的速率下调，其中α-PKC下调最快，ε-PKC最慢。我们利用α-PKC、ε-PKC和δ-PKC的选择性下调来研究这些同工型在新生心肌细胞中PKC磷酸化依赖性事件中的作用。我们的研究结果表明，ε-PKC负责去氧肾上腺素诱导的内源性PKC特异性底物MARCKS的磷酸化。相比之下，激动剂诱导的c-fos表达不太可能由ε-PKC介导，因为该反应迅速下调且明显依赖于钙。我们发现PKC同工型对神经激素的反应不同，这表明它们在心脏功能中发挥着独特而特定的作用。
