Pachter J A, Mayer-Ezell R, Cleven R M, Fawzi A B
Schering-Plough Research Institute, Kenilworth, NJ 07033-0539.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):153-8. doi: 10.1042/bj2940153.
To evaluate the role of protein kinase C (PKC) in regulation of cellular responsiveness to mitogens, we used rat 6 (R6) fibroblasts that stably overexpress the beta 1 isoenzyme of protein kinase C (PKC-beta 1). The potent vasoconstrictor and mitogen endothelin-1 (ET-1; 100 nM) was substantially more effective in stimulating InsP3 accumulation in PKC-beta 1-overexpressing fibroblasts (PKC3 cells) than in control fibroblasts lacking the PKC-beta 1 cDNA insert. PKC3 cells were found to express a 7-fold greater number of endothelin receptors than did control cells, whereas both cell lines showed equivalent Kd values. These receptors were of the ETA subtype, as defined by a 1000-fold greater affinity for ET-1 than for ET-3. Changes in intracellular free Ca2+ levels ([Ca2+]i) in response to ET-1 measured with the fluorescent Ca2+ indicator fura-2 showed that ET-1 was more potent and efficacious in stimulating [Ca2+]i in PKC3 cells than in control fibroblasts. The ET-1-induced Ca2+ rise was completely blocked by the selective ETA antagonist BQ123, but only slightly diminished by extracellular application of 2 mM EGTA. In contrast with the effects of PKC-beta 1 overexpression on responsiveness to ET-1, alpha-thrombin, which was previously found to have a weaker effect on InsP3 accumulation in PKC-beta 1-overexpressing cells, was also a less effective stimulator of [Ca2+]i in PKC3 cells than in control cells. These results demonstrate that, although the Ca2+ response to alpha-thrombin is diminished by PKC-beta 1 overexpression, ETA receptor number and cellular responsiveness to ET-1 are increased in PKC-beta 1-overexpressing cells.
为了评估蛋白激酶C（PKC）在调节细胞对有丝分裂原反应性中的作用，我们使用了稳定过表达蛋白激酶Cβ1同工酶（PKC-β1）的大鼠6（R6）成纤维细胞。强效血管收缩剂和有丝分裂原内皮素-1（ET-1；100 nM）在刺激过表达PKC-β1的成纤维细胞（PKC3细胞）中肌醇三磷酸（InsP3）积累方面，比在缺乏PKC-β1 cDNA插入片段的对照成纤维细胞中显著更有效。发现PKC3细胞表达的内皮素受体数量比对照细胞多7倍，而两种细胞系显示出相同的解离常数（Kd）值。这些受体属于ETA亚型，其对ET-1的亲和力比对ET-3高1000倍。用荧光钙指示剂fura-2测量的对ET-1反应时细胞内游离钙离子水平（[Ca2+]i）的变化表明，ET-1在刺激PKC3细胞中的[Ca2+]i方面比在对照成纤维细胞中更有效且更具效力。ET-1诱导的钙离子升高被选择性ETA拮抗剂BQ123完全阻断，但通过细胞外施加2 mM乙二醇双四乙酸（EGTA）仅略有减弱。与PKC-β1过表达对ET-1反应性的影响相反，先前发现α-凝血酶在过表达PKC-β1的细胞中对InsP3积累的作用较弱，在PKC3细胞中它对[Ca2+]i的刺激作用也比在对照细胞中更弱。这些结果表明，虽然PKC-β1过表达会降低对α-凝血酶的钙离子反应，但在过表达PKC-β1的细胞中ETA受体数量和细胞对ET-1的反应性会增加。
