ROLE OF PHOSPHOLIPASE A(2) 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 si gnaling cascade. We investigated the regulation and differential stimu lation of PLA(2)s by comparing die PLA(2) activities associated with t he membranes and cytosol of rabbit renal proximal tubular epithelial c ells after stimulation with angiotensin II, epidermal growth factor, a nd bradykinin. Both fractions demonstrated PLA(2) activity that was di thiothreitol insensitive, required micromolar concentrations of Ca2+ f or optimal activity, and was inhibited in a dose-dependent manner by a n antiserum to a cytosolic PLA(2) with a molecular mass of 85 kD. Howe ver, membrane-associated PLA(2) did not demonstrate significant substr ate specificity, whereas 1-steroyl-2-[C-14]arachidonylphosphatidyl cho line was the preferred substrate for cPLA(2). An antiserum generated a gainst mastoparan, a known PLA(2) activator, inhibited membrane-but no t cytosol-associated PLA(2) activity. Membrane fractions showed a broa d pH range (7.5 to 8.5) for optimal PLA(2) activity, whereas cytosol w as maximum at pH 9.5. Angiotensin II stimulated membrane-associated PL A(2) activity by 88%, whereas bradykinin and epidermal growth factor i nhibited activity by 54% and 41%, respectively. The three agonists sti mulated cPLA(2). Moreover, angiotensin II-induced activation of membra ne-associated PLA(2) preceded the activation of cPLA(2). These results demonstrate differential localization and regulation of proximal tubu lar epithelial PLA-isozymes, which may determine the pattern of subseq uent arachidonic acid metabolism by the cytochrome P450 system.