Horwitz J, Ricanati S
Department of Pediatrics, University of Chicago, Illinois.
J Neurochem. 1992 Oct;59(4):1474-80. doi: 10.1111/j.1471-4159.1992.tb08463.x.
Bradykinin is known to activate phospholipase D in PC12 cells. Because bradykinin may also activate protein kinase C in these cells, the possible role of this kinase in mediating the action of bradykinin was investigated. Phospholipase D activity in PC12 cells was assayed by measuring the formation of [3H]phosphatidylethanol in cells prelabeled with [3H]palmitic acid and incubated in the presence of ethanol. The phorbol ester phorbol dibutyrate mimicked the effect of bradykinin on [3H]phosphatidylethanol formation. The protein kinase C inhibitor staurosporine (1 microM) significantly attenuated the effect of phorbol dibutyrate (35-70%) but did not block bradykinin-stimulated [3H]phosphatidylethanol formation. In addition, the effect of phorbol dibutyrate was additive with that of bradykinin. Prolonged treatment of PC12 cells with phorbol dibutyrate (24 h), which depletes cells of protein kinase C, greatly attenuated bradykinin-stimulated [3H]phosphatidylethanol accumulation in intact cells. This treatment caused a 55% decrease in both fluoride-stimulated [3H]phosphatidylethanol production in the intact cell and phospholipase D activity as assessed by an in vitro assay using an exogenous substrate. Therefore, the effect of prolonged phorbol dibutyrate pretreatment on bradykinin-stimulated [3H]phosphatidylethanol production could not be attributed exclusively to the depletion of protein kinase C. Thus, although the data with phorbol ester suggest that activation of protein kinase C leads to an increase in phospholipase D activity, this kinase probably does not play a role in mediating the effect of bradykinin. Finally, although pretreatment with phorbol dibutyrate completely blocked bradykinin-stimulated [3H]phosphatidylethanol production in the intact cell, it only partially (approximately 50%) inhibited bradykinin-stimulated [3H]diacylglycerol formation.(ABSTRACT TRUNCATED AT 250 WORDS)
已知缓激肽可激活PC12细胞中的磷脂酶D。由于缓激肽也可能激活这些细胞中的蛋白激酶C，因此研究了该激酶在介导缓激肽作用中的可能作用。通过测量用[3H]棕榈酸预标记并在乙醇存在下孵育的细胞中[3H]磷脂酰乙醇的形成来测定PC12细胞中的磷脂酶D活性。佛波酯佛波醇二丁酸酯模拟了缓激肽对[3H]磷脂酰乙醇形成的作用。蛋白激酶C抑制剂星形孢菌素（1 microM）显著减弱了佛波醇二丁酸酯的作用（35 - 70%），但并未阻断缓激肽刺激的[3H]磷脂酰乙醇形成。此外，佛波醇二丁酸酯的作用与缓激肽的作用具有加和性。用佛波醇二丁酸酯（24小时）对PC12细胞进行长时间处理，可耗尽细胞中的蛋白激酶C，这大大减弱了完整细胞中缓激肽刺激的[3H]磷脂酰乙醇积累。这种处理使完整细胞中氟化物刺激的[3H]磷脂酰乙醇产生以及通过使用外源底物的体外测定评估的磷脂酶D活性均降低了55%。因此，佛波醇二丁酸酯长时间预处理对缓激肽刺激的[3H]磷脂酰乙醇产生的影响不能完全归因于蛋白激酶C的耗尽。因此，尽管佛波酯的数据表明蛋白激酶C的激活导致磷脂酶D活性增加，但该激酶可能在介导缓激肽的作用中不起作用。最后，尽管用佛波醇二丁酸酯预处理完全阻断了完整细胞中缓激肽刺激的[3H]磷脂酰乙醇产生，但它仅部分（约50%）抑制了缓激肽刺激的[3H]二酰基甘油形成。（摘要截短至250字）
