The role of Phospholipase A2 in Calcium-Ionophore-Mediated Injury to Rat Gastric Mucosal Cells

Abstract

Although transient increases in intracellularCa²⁺ ([Ca²⁺]_i) underliea number of important physiological processes, sustainedelevations in [Ca²⁺]_i mediatedamage to a number of tissues and cell types including gastric mucosal cells. Increases in[Ca²⁺]_i can activate phospholipidhydrolysis via increases in phospholipase A₂(PLA₂) activity and subsequent cell injury.In the present study we have examined wheThe r [Ca²⁺]_i-inducedgastric cellular injury is mediated by PLA₂activation. Gastric mucosal cells were harvested fromrat stomachs after pronase digestion. Cell integrity wasassessed using trypan blue dye exclusion and release of lysozomal enzymes.PLA₂ activity was estimated colorimetricallyby determination of thiol release from the substrate,arachidonyl thio-PC. In these studies calcium ionophore A23187 (3-25 μM) resulted in an increase incell injury. The damage produced by A23187 (12.5 μM)was inhibited by preincubation of cells with thePLA₂ inhibitor, quinacrine (1-100 μM).Quinacrine did not reduce ethanol (10% w/v) mediated-celldamage. Similarly Ca²⁺ ionophore A23187treatment resulted in a concentration-dependent increasein PLA₂ activity in gastric cells. Theincrease in PLA₂ activity was attenuated if cells were incubated inCa²⁺-depleted medium containing EGTA (4 mM).FurThe rmore lysophospholipids generated byPLA₂ (lysophosphatidylethanolamine andlysophosphatidylcholine; 100 μM) also increased the degree of cell injury.Pretreatment of cells with the PAF antagonist WEB 2086(10^-6 and 10^-5 M), the leukotrienesynthase inhibitor 5,6-dehydroarachidonic acid (10μM), or the thromboxane synthase inhibitor furegrelate (1 μM)decrease A23187-mediated cell injury. These data suggestthat Ca²⁺ ionophore-mediated increases in[Ca²⁺]_i result in gastric cellinjury and this effect is mediated in part by PLA₂ activation andsubsequent release of free fatty acids andlysophosphatides.