Role of PLA(2), PLC, and PLD in bradykinin-induced release of arachidonic acid in MDCK cells

The role of cytosolic phospholipase A(2) (cPLA(2)), phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the bradykinin (BK)-stimulated release of arachidonic acid (AA) was examined in Madin-Darby canine kidney (MDCK) cells. Release of AA, phosphorylcholine, choline, and phosphatidic acid (PA) or the transphosphatidylation product, phosphatidylethanol, was detected after 1 min of BK stimulation. A role for PC-PLC was confirmed with D609, which reduced BK-stimulated AA by 70%. Ethanol (EtOH), which blunts PA formation, diminished BK-stimulated AA release by 50%. Together, D609 and EtOH inhibited this release almost completely. Evidence indicated that diacylglycerol and PA can enhance PLA(2) activity when added to cytosol extracts. The enzyme responsible for AA release was characterized as cPLA(2), since PLA(2) activity assayed in cell extracts was largely inhibited by an antibody to this enzyme. The membrane fraction PLA(2) activity increased significantly in BK-stimulated cells. We conclude that BK signaling in MDCK cells is mediated by the lipid products of PC-PLC and PLD, increasing cPLA(2) activity, possibly by causing perturbations in the bilayer structure of its substrate, by a direct effect on the enzyme or by activation of protein kinases such as protein kinase C.