Angiotensin II Type 2 Receptor Subtype Mediates Phospholipase A₂–Dependent Signaling in Rabbit Proximal Tubular Epithelial Cells

We investigated the ability of angiotensin II (Ang II) or the stable analogue [Sar1]-Ang II to increase intracellular and extracellular free arachidonic acid in primary cultures of rabbit proximal tubular epithelial cells to better characterize the receptor subtype and orientation of phospholipase A2 (PLA2)-mediated signaling. Proximal tubular cells were labeled with [3H]arachidonic acid for 4 hours and then treated with Ang II or [Sar1]-Ang II. Lipids were extracted from labeled cells, separated by thin-layer chromatography, and quantified by liquid scintillation counting. Ang II (10 mumol/L, 1 minute) stimulated an increase in intracellular free [3H]arachidonic acid from 21.0 +/- 2.0 to 32.2 +/- 2.8 disintegrations per minute/microgram protein, an effect that was potentiated by EGTA. [Sar1]-Ang II stimulated a time- and concentration-dependent increase in [3H]arachidonic acid release from labeled cells. Release of [3H]arachidonic acid was maximal at 10 mumol/L [Sar1]-Ang II, with an EC50 of approximately 3 mumol/L. Ang II receptor antagonists caused concentration-dependent inhibition of [Sar1]-Ang II-stimulated [3H]arachidonic acid release with the following order of potency: CGP 42112 = PD 123319 > losartan. Furthermore, in proximal tubular epithelial cells grown on polyester membrane filters, the Ang II receptor that mediated arachidonic acid release was predominantly apical rather than basolateral. These observations are consistent with activation of a Ca(2+)-independent, apical PLA2 isoform in epithelial cells through an Ang II type 2 receptor subtype.