Characterization and regulation of cytosolic phospholipase A(2) activity following bradykinin stimulation of rabbit cortical collecting duct cells.

Abstract

The mammalian kidney, in particular the collecting duct segment of the nephron, is a major site of renal sodium and water reabsorption. One of the many agonists known to regulate collecting duct function is bradykinin (BK), a nonapeptide which exerts natriuretic and diuretic effects through its ability to increase arachidonic acid (AA) availability and prostaglandin production. Evidence presented herein indicates that 85 kDa cytosolic phospholipase A$\sb2$ (cPLA$\sb2)$ is the enzyme accountable for BK-mediated AA. This is based on the following observations: RCCD cells express cPLA$\sb2$ protein, an arachidonyl trifluoromethyl ketone inhibitor of cPLA$\sb2$ completely blunted BK-stimulated AA release, both basal and BK-stimulated PLA$\sb2$ activities were significantly diminished following treatment with an antibody to this enzyme, and finally, PLA$\sb2$ in vitro activity could be stimulated by submillimolar calcium concentrations and was dithiothreitol (DTT)-insensitive. Strategies were also employed to reveal the involvement of protein kinase C (PKC) in BK-mediated AA release, cPLA$\sb2$ activation and cPLA$\sb2$ phosphorylation. Chemical inhibition of PKC reduced both BK-stimulated AA release and cPLA$\sb2$ activity; however, BK-stimulated AA release appeared limited by calcium mobilization rather than by PKC. Cytosolic PLA$\sb2$ activity could be stimulated by phorbol ester (PMA) and the magnitude of this activation was similar to that caused by BK. A direct correlation between the level of cPLA$\sb2$ phosphorylation and activation was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated responses. Furthermore, the increase in cPLA$\sb2$ phosphorylation was found to occur on serine residues. (Abstract shortened by UMI.)