title: Role of phospholipase A2 isozymes in agonist-mediated signaling in proximal tubular epithelium.
abstract: Angiotensin II in proximal tubule epithelium is known to stimulate the release of arachidonic acid after stimulation of phospholipase A2 (PLA2) 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 PLA2s by comparing the PLA2 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 PLA2 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 PLA2 with a molecular mass of 85 kD. However, membrane-associated PLA2 did not demonstrate significant substrate specificity, whereas 1-steroyl-2-[14C]arachidonylphosphatidyl choline was the preferred substrate for cPLA2. An antiserum generated against mastoparan, a known PLA2 activator, inhibited membrane- but not cytosol-associated PLA2 activity. Membrane fractions showed a broad pH range (7.5 to 8.5) for optimal PLA2 activity, whereas cytosol was maximum at pH 9.5. Angiotensin II stimulated membrane-associated PLA2 activity by 88%, whereas bradykinin and epidermal growth factor inhibited activity by 54% and 41%, respectively. The three agonists stimulated cPLA2. Moreover, angiotensin II-induced activation of membrane-associated PLA2 preceded the activation of cPLA2. These results demonstrate differential localization and regulation of proximal tubular epithelial PLA2 isozymes, which may determine the pattern of subsequent arachidonic acid metabolism by the cytochrome P450 system.
authors.affiliation: Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Ohio 44106, USA.
authors.forename: S
authors.initials: S
authors.lastname: Harwalkar
authors.forename: C H
authors.initials: CH
authors.lastname: Chang
authors.forename: N O
authors.initials: NO
authors.lastname: Dulin
authors.forename: J G
authors.initials: JG
authors.lastname: Douglas
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007527:Isoenzymes / Q000032:analysis / Q000502:physiology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D008566:Membranes / Q000201:enzymology / Q000378:metabolism; D010741:Phospholipases A / Q000032:analysis / Q000502:physiology*; D054467:Phospholipases A2; D011817:Rabbits; D015398:Signal Transduction; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D007527:Isoenzymes; D000804:Angiotensin II; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium