The role of phospholipase A(2) in calcium-ionophore-mediated injury to ratgastric mucosal cells

Although transient increases in intracellular Ca2+ ([Ca2+](i)) underlie a n umber of important physiological processes, sustained elevations in [Ca2+]( i) mediate damage to a number of tissues and cell types including gastric m ucosal cells. Increases in [Ca2+](i) can activate phospholipid hydrolysis v ia increases in phospholipase A(2) (PLA(2)) activity and subsequent cell in jury. In the present study we have examined whether [Ca2+](i)-induced gastr ic cellular injury is mediated by PLA(2) activation. Gastric mucosal cells were harvested from rat stomachs after pronase digestion. Cell integrity wa s assessed using trypan blue dye exclusion and release of lysozomal enzymes . PLA(2) activity was estimated colorimetrically by determination of thiol release from the substrate, arachidonyl thio-PC. In these studies calcium i onophore A23187 (3-25 mu M) resulted in an increase in cell injury. The dam age produced by A23187 (12.5 mu M) was inhibited by preincubation of cells with the PLA(2) inhibitor, quinacrine (1-100 mu M). Quinacrine did not redu ce ethanol (10% w/v) mediated-cell damage. Similarly Ca2+ ionophore A23187 treatment resulted in a concentration-dependent increase in PLA, activity i n gastric cells. The increase in PLA(2) activity was attenuated if cells we re incubated in Ca2+-depleted medium containing EGTA (4 mM). Furthermore ly sophospholipids generated by PLA, (lysophosphatidylethanolamine and lysopho sphatidylcholine; 100 mu M) also increased the degree of cell injury. Pretr eatment of cells with the PBF antagonist WEB 2086 (10(-6) and 10(-5) M), th e leukotriene synthase inhibitor 5,6-dehydroarachidonic acid (10 mu M), or the thromboxane synthase inhibitor furegrelate (1 mu M) decrease A23187-med iated cell injury, These data suggest that Ca2+ ionophore-mediated increase s in [Ca2+](i) result in gastric cell injury and this effect is mediated in part by PLA(2) activation and subsequent release of free fatty acids and l ysophosphatides.