Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes

We characterized phospholipase A(2) (PLA(2)) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca2+ dependency. Membrane-associated PLA(2) was found to be an order of magnitude greater than cytosolic PLA(2). Ventricular myocyte PLA(2) activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca2+-independent PLA(2) from Chinese hamster ovary cells recognized a membrane-associated protein with a molecular mass of approximately 80 kDa; however, differences in pH optima, response to inhibitors, and substrate selectivity of membrane-associated and cytosolic PLA(2) activity suggest the presence of multiple Ca2+-independent PLA(2). Pretreatment with bromoenol lactone, a specific inhibitor of Ca2+-independent PLA(2), significantly attenuated membrane-associated and cytosolic PLA(2) in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachiodnyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA(2) activity under all conditions tested. Ventricular myocyte PLA(2) activity was significantly inhibited by ATP, GTP, and their nonhydrolyzable analogs and was regulated by protein kinase C activity. These studies demonstrate the presence of one or more unique membrane-associated Ca2+-independent PLA(2) in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.