BRADYKININ-STIMULATED CPLA(2) PHOSPHORYLATION IS PROTEIN-KINASE-C DEPENDENT IN RABBIT CCD CELLS

We have used an established cell line of rabbit cortical collecting du ct (RCCD) epithelial cells representing a mixed population of principa l and intercalated cell types to determine which phospholipase A(2) (P LA(2)) enzyme therein is responsible for bradykinin (BK)-stimulated ar achidonic acid (AA) release and how its activation is regulated. BK-st imulated AA release was reduced 92% by arachidonyl trifluoromethyl ket one, an inhibitor of cytosolic PLA(2) (cPLA(2)). Examination of PLA(2) activity in vitro demonstrated that BK stimulation resulted in a grea ter than twofold increase in PLA(2) activity and that this activity wa s dithiothreitol insensitive and was inhibited by an antibody directed against cPLA(2). To determine a possible role for protein kinase C (P KC) in the BK-mediated activation of cPLA(2), we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA(2) a ctivity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA relea se and cPLA(2) activity by 51 and 58%, respectively. cPLA(2) activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] di splayed a similar degree of activation and was associated with an incr ease in serine phosphorylation identical to that caused by BK. The pho sphorylation-induced activation of this enzyme was confirmed by the ph osphatase-mediated reversal of both BK- and PMA-stimulated cPLA(2) act ivity. In addition, we have also found that PMA stimulation did not ca use a synergistic potentiation of BK-stimulated AA release as did calc ium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken to gether, indicate that cPLA(2) is the enzyme responsible for BK-mediate d AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA(2) to BK.