Calcium-independent phospholipase A(2) in isolated rabbit ventricular myocytes

We characterized phospholipase A(2) (PLA(2)) activity in isolated rabbit ve ntricular myocytes with respect to subcellular distribution, substrate spec ificity, 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. I mmunoblot analysis using an antibody to cytosolic Ca2+-independent PLA(2) f rom Chinese hamster ovary cells recognized a membrane-associated protein wi th a molecular mass of approximately 80 kDa; however, differences in pH opt ima, response to inhibitors, and substrate selectivity of membrane-associat ed and cytosolic PLA(2) activity suggest the presence of multiple Ca2+-inde pendent PLA(2). Pretreatment with bromoenol lactone, a specific inhibitor o f Ca2+-independent PLA(2), significantly attenuated membrane-associated and cytosolic PLA(2) in unstimulated and thrombin-stimulated myocytes. Pretrea tment with methyl arachiodnyl fluorophosphonate, mepacrine, or dibucaine ha d no significant effect on PLA(2) activity under all conditions tested. Ven tricular myocyte PLA(2) activity was significantly inhibited by ATP, GTP, a nd their nonhydrolyzable analogs and was regulated by protein kinase C acti vity. These studies demonstrate the presence of one or more unique membrane -associated Ca2+-independent PLA(2) in isolated ventricular myocytes that e xhibit a preference for phospholipids with arachidonate at the sn-2 positio n and that are activated by thrombin stimulation.