Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A(2) in human erythrocytes

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). A nalysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatid ylethanolamine during incubation with ionomycin and sPLA(2). Several possib le mechanisms for the effect of ionomycin were considered. Involvement of i ntracellular phospholipases A, was excluded since inhibitors of these enzym es had no effect. Assessment of membrane oxidation by cis-parinaric acid fl uorescence and comparison to the oxidants diamide and phenylhydrazine revea led that oxidation does not participate in the effect of iono- mycin. Incub ation with ionomycin caused classical physical changes to the erythrocyte m embrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of mi crovesicles. Experiments with phenylhydrazine, KCI, quinine, merocyanine 54 0, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required t o induce susceptibility. Results from fluorescence spectroscopy and two-pho ton excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membr ane lipids.