Weiss B A, Slivka S R, Insel P A
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Mol Pharmacol. 1989 Aug;36(2):317-26.
Madin-Darby canine kidney cells (MDCK) are known to release free arachidonic acid and arachidonic acid metabolites (AA) in response to tumor-promoting phorbol esters, such as tetradecanoyl phorbol-13-acetate, and to agonists active at alpha 1-adrenergic and bradykinin B2 receptors. These experiments were conducted to define the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) activation in the stimulation of AA release, in the clonal isolate cell line MDCK-D1, by use of three inhibitors of protein kinase C, sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine. We found that alpha 1-adrenergic- and bradykinin-stimulated [3H]AA release can be distinguished by differential dependence on protein kinase C; epinephrine-stimulated release was more dependent on protein kinase C activation than was bradykinin-stimulated release. The inhibition of bradykinin-stimulated AA release by sphingosine (20.2 +/- 6.1%) was substantially less than the inhibition observed for tetradecanoyl phorbol-13-acetate- (67.2 +/- 5.5%) and epinephrine-stimulated release (50.2 +/- 9.2%). These findings were confirmed by results using H-7 and staurosporine. The relative independence of bradykinin-stimulated AA release of protein kinase C was also demonstrated by the inability of phorbol ester-induced down-regulation of protein kinase C to eliminate bradykinin-stimulated AA release. The inhibition of alpha 1-adrenergic receptor-mediated AA release by sphingosine, H-7, and staurosporine was not due to a change in receptor number or affinity. Analysis of the products comprising [3H]AA release indicated that treatment with sphingosine did not change the composition of the released AA (34-48% prostaglandin E2, 17-27% free arachidonic acid, and 25-51% unidentified metabolites). These results indicate that two different types of hormone receptors in the same cell type can promote AA release by mechanisms that differ in their dependence on protein kinase C. The protein kinase C-dependent mechanism may represent protein kinase C-mediated activation of phospholipase A2.
已知马-达二氏犬肾细胞（MDCK）会响应促肿瘤佛波酯（如十四酰佛波醇-13-乙酸酯）以及对α1-肾上腺素能受体和缓激肽B2受体有活性的激动剂，释放游离花生四烯酸和花生四烯酸代谢物（AA）。本实验旨在通过使用三种蛋白激酶C抑制剂（鞘氨醇、1-（5-异喹啉磺酰基）-2-甲基哌嗪（H-7）和星形孢菌素）来确定Ca2+/磷脂依赖性蛋白激酶（蛋白激酶C）激活在克隆分离细胞系MDCK-D1中刺激AA释放过程中的作用。我们发现，α1-肾上腺素能受体和缓激肽刺激的[3H]AA释放可通过对蛋白激酶C的不同依赖性来区分；肾上腺素刺激的释放比缓激肽刺激的释放更依赖蛋白激酶C的激活。鞘氨醇对缓激肽刺激的AA释放的抑制作用（20.2±6.1%）明显小于对十四酰佛波醇-13-乙酸酯刺激的释放（67.2±5.5%）和肾上腺素刺激的释放（50.2±9.2%）的抑制作用。使用H-7和星形孢菌素得到的结果证实了这些发现。佛波酯诱导的蛋白激酶C下调无法消除缓激肽刺激的AA释放，这也证明了缓激肽刺激的AA释放对蛋白激酶C的相对独立性。鞘氨醇、H-7和星形孢菌素对α1-肾上腺素能受体介导的AA释放的抑制作用并非由于受体数量或亲和力的改变。对构成[3H]AA释放的产物的分析表明，用鞘氨醇处理不会改变释放的AA的组成（34 - 48%为前列腺素E2，17 - 27%为游离花生四烯酸，25 - 51%为未鉴定的代谢物）。这些结果表明，同一细胞类型中的两种不同类型的激素受体可通过对蛋白激酶C依赖性不同的机制促进AA释放。蛋白激酶C依赖性机制可能代表蛋白激酶C介导的磷脂酶A2的激活。
