title: Bradykinin-stimulated cPLA2 phosphorylation is protein kinase C dependent in rabbit CCD cells.
abstract: We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2 (PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2 (cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
authors.forename: M A
authors.initials: MA
authors.lastname: Lal
authors.forename: C R
authors.initials: CR
authors.lastname: Kennedy
authors.forename: P R
authors.initials: PR
authors.lastname: Proulx
authors.forename: R L
authors.initials: RL
authors.lastname: Hébert
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D003512:Cyclohexanones / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007672:Kidney Cortex / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D003512:Cyclohexanones; D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; C081565:arachidonyltrifluoromethane; D016718:Arachidonic Acid; D000001:Calcimycin; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin