Bradykinin and phorbol dibutyrate activate phospholipase D in PC12 cells by different mechanisms.

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)