Lamers J M, Eskildsen-Helmond Y E, Resink A M, de Jonge H W, Bezstarosti K, Sharma H S, van Heugten H A
Department of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands.
J Cardiovasc Pharmacol. 1995;26 Suppl 3:S100-3.
We have previously demonstrated that stimulation of cultured rat neonatal cardiomyocytes by endothelin-1 (ET-1) induces rapid activation of phospholipase C-beta (PLC-beta), accompanied by transient expression of proto-oncogenes and subsequent development of hypertrophy and characteristic phenotypic changes. In the present study we examined the ET-1-induced hypertrophic response in relation to the initial signaling by phospholipase D (PLD) and protein kinase C (PKC). ET-1 (10(-8) M) induced hypertrophy after 48 h, as judged by protein/DNA ratio. The formation (0.5 h) of 14C-labeled phosphatidylethanol ([14C]PEth) in the presence of exogenous ethanol (0.5%) in [14C]palmitate prelabeled cells, which reflects the PLD activity, was increased 1.9- and 5.6-fold by ET-1 and phorbolester (PMA, 10(-6) M), respectively. The translocation of PKC isoforms from the cytosol to the membrane fraction was examined by immunoblot analysis using specific antibodies for PKC-alpha and -epsilon. ET-1 caused a rapid (within 15 s) and sustained disappearance of PKC-epsilon but not of PKC-alpha, from the cytosol. The translocation of PKC-epsilon to the membrane fraction was just detectable. However, PMA (10(-7) M) showed a rapid, sustained, and clearly detectable translocation of PKC-alpha and PKC-epsilon. The results indicate that the ET-1-induced development of hypertrophy via activation of distinct PKC isoenzymes may be initiated not only by PLC-beta but also by PLD signaling.
我们之前已经证明，内皮素-1（ET-1）刺激培养的新生大鼠心肌细胞可诱导磷脂酶C-β（PLC-β）迅速激活，同时伴有原癌基因的瞬时表达以及随后的肥大发展和特征性表型变化。在本研究中，我们研究了ET-1诱导的肥大反应与磷脂酶D（PLD）和蛋白激酶C（PKC）的初始信号传导之间的关系。通过蛋白质/DNA比率判断，ET-1（10^(-8) M）在48小时后诱导肥大。在[14C]棕榈酸预标记的细胞中加入外源性乙醇（0.5%）时，14C标记的磷脂酰乙醇（[14C]PEth）的形成（0.5小时）反映了PLD活性，ET-1和佛波酯（PMA，10^(-6) M）分别使其增加了1.9倍和5.6倍。使用针对PKC-α和-ε的特异性抗体，通过免疫印迹分析检测PKC同工型从胞质溶胶向膜部分的转位。ET-1导致PKC-ε从胞质溶胶中迅速（15秒内）且持续消失，但PKC-α没有。PKC-ε向膜部分的转位刚刚可检测到。然而，PMA（10^(-7) M）显示PKC-α和PKC-ε有快速、持续且明显可检测到的转位。结果表明，ET-1通过激活不同的PKC同工酶诱导肥大的发展可能不仅由PLC-β启动，也由PLD信号传导启动。
