Ma. Lal et al., BRADYKININ-STIMULATED CPLA(2) PHOSPHORYLATION IS PROTEIN-KINASE-C DEPENDENT IN RABBIT CCD CELLS, American journal of physiology. Renal, fluid and electrolyte physiology, 42(6), 1997, pp. 907-915
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.