Role of phospholipase A2 isozymes in agonist-mediated signaling in proximal tubular epithelium

Angiotensin II in proximal tubule epithelium is known to stimulate the release of arachidonic acid after stimulation of phospholipase A(2) (PLA(2)) independent of phospholipase C-mediated signaling. Furthermore, an angiotensin II type 2 receptor subtype has been linked to this signaling cascade. We investigated the regulation and differential stimulation of PLA(2)s by comparing die PLA(2) activities associated with the membranes and cytosol of rabbit renal proximal tubular epithelial cells after stimulation with angiotensin II, epidermal growth factor, and bradykinin. Both fractions demonstrated PLA(2) activity that was dithiothreitol insensitive, required micromolar concentrations of Ca2+ for optimal activity, and was inhibited in a dose-dependent manner by an antiserum to a cytosolic PLA(2) with a molecular mass of 85 kD. However, membrane-associated PLA(2) did not demonstrate significant substrate specificity, whereas 1-steroyl-2-[C-14]arachidonylphosphatidyl choline was the preferred substrate for cPLA(2). An antiserum generated against mastoparan, a known PLA(2) activator, inhibited membrane-but not cytosol-associated PLA(2) activity. Membrane fractions showed a broad pH range (7.5 to 8.5) for optimal PLA(2) activity, whereas cytosol was maximum at pH 9.5. Angiotensin II stimulated membrane-associated PLA(2) activity by 88%, whereas bradykinin and epidermal growth factor inhibited activity by 54% and 41%, respectively. The three agonists stimulated cPLA(2). Moreover, angiotensin II-induced activation of membrane-associated PLA(2) preceded the activation of cPLA(2). These results demonstrate differential localization and regulation of proximal tubular epithelial PLA-isozymes, which may determine the pattern of subsequent arachidonic acid metabolism by the cytochrome P450 system.