Slivka S R, Insel P A
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1988 Oct 15;263(29):14640-7.
Many types of peptide hormone and neurotransmitter receptors mediate hydrolysis of phosphoinositides (PI) and arachidonic acid and arachidonic acid metabolite (AA) release, but the relation between these responses is not clearly defined. We have characterized bradykinin (BK)-mediated AA release and PI hydrolysis in clonal Madin-Darby canine kidney cells (MDCK-D1). Both responses occurred over a similar dose range in response to the B1 and B2 receptor agonist, BK, but not in response to the B1 receptor-selective agonist des-Arg-BK. To test whether AA release occurs via a mechanism which is sequential to and dependent upon PI hydrolysis, we used the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C. TPA treatment blocked BK-mediated PI hydrolysis in MDCK-D1 cells, while at the same time and at similar concentrations enhancing BK-mediated AA release. Thus, TPA treatment dissociated BK-mediated AA release from PI hydrolysis. In addition, treatment of MDCK-D1 cells with neomycin blocked BK-mediated hydrolysis of phosphatidylinositol bisphosphate without reducing BK-mediated AA release. BK treatment increased formation of lysophospholipids with a time course in accord with BK-mediated AA release, indicating that at least part of the BK-mediated AA release was likely derived from activation of phospholipase A2. BK-mediated lysophospholipid production was enhanced by pretreatment with TPA, suggesting that the mechanism of AA release before and after treatment with TPA was the same. BK-mediated AA release and lysophospholipid production was dependent on the presence of extracellular calcium, while the enhanced responses to BK in the presence of TPA were not dependent on the presence of extracellular calcium. TPA treatment also enhanced AA release and lysophospholipid production in response to the calcium ionophore A23187. From these data we propose that BK, acting at B2 receptors, promotes AA release in MDCK cells via a mechanism which is 1) independent of polyphosphoinositide hydrolysis by phospholipase C, 2) dependent upon influx of extracellular calcium and activation of phospholipase A2, and 3) enhanced by activation of protein kinase C.
许多类型的肽类激素和神经递质受体介导磷酸肌醇（PI）的水解以及花生四烯酸和花生四烯酸代谢产物（AA）的释放，但这些反应之间的关系尚未明确界定。我们已对克隆的麦氏犬肾细胞（MDCK-D1）中缓激肽（BK）介导的AA释放和PI水解进行了表征。这两种反应在对B1和B2受体激动剂BK的相似剂量范围内发生，但对B1受体选择性激动剂去精氨酸缓激肽（des-Arg-BK）无反应。为了测试AA释放是否通过一种依次发生且依赖于PI水解的机制发生，我们使用了佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA），它可激活蛋白激酶C。TPA处理阻断了MDCK-D1细胞中BK介导的PI水解，而与此同时且在相似浓度下增强了BK介导的AA释放。因此，TPA处理使BK介导的AA释放与PI水解分离。此外，用新霉素处理MDCK-D1细胞可阻断BK介导的磷脂酰肌醇二磷酸水解，而不会降低BK介导的AA释放。BK处理增加了溶血磷脂的形成，其时间进程与BK介导的AA释放一致，表明BK介导的AA释放至少部分可能源自磷脂酶A2的激活。TPA预处理增强了BK介导的溶血磷脂生成，表明TPA处理前后AA释放的机制相同。BK介导的AA释放和溶血磷脂生成依赖于细胞外钙的存在，而在TPA存在下对BK增强的反应不依赖于细胞外钙的存在。TPA处理还增强了对钙离子载体A23187的反应中AA释放和溶血磷脂生成。根据这些数据，我们提出，作用于B2受体的BK通过以下机制促进MDCK细胞中AA释放：1）独立于磷脂酶C介导的多磷酸肌醇水解；2）依赖于细胞外钙内流和磷脂酶A2的激活；3）通过蛋白激酶C的激活而增强。
