Calcium-independent phospholipase A₂ in isolated rabbit ventricular myocytes

We characterized phospholipase A₂ (PLA₂) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca²⁺ dependency. Membrane-associated PLA₂ was found to be an order of magnitude greater than cytosolic PLA₂. Ventricular myocyte PLA₂ activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca²⁺independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca²⁺-independent PLA₂ 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 PLA₂ activity suggest the presence of multiple Ca²⁺-independent PLA₂. Pretreatment with bromoenol lactone, a specific inhibitor of Ca²⁺-independent PLA₂, significantly attenuated membrane- associated and cytosolic PLA₂ in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachidonyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA₂ activity under all conditions tested. Ventricular myocyte PLA₂ 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 PLA₂ in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.