Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A 2 (sPLA 2 ). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA 2 . Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A 2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA 2 is a consequence of increased order of membrane lipids.Under normal conditions, healthy cell membranes resist catalysis by secretory phospholipase A 2 (sPLA 2 ) 1 (1-4). However, they may become susceptible under circumstances that cause alteration of membrane physical properties (1-4). Previous studies using artificial membranes demonstrated that alterations that increase susceptibility generally increase the anionic charge of the outer leaflet, increase bilayer curvature, and/or decrease interactions among neighboring phospholipids (5-9). In some cases, enhanced susceptibility of artificial membranes depends on an increase in the order of the phospholipids (8, 10 -14). These changes increase susceptibility by augmenting the binding of sPLA 2 and/or by improving access of membrane phospholipids to the active site of the enzyme (5-12, 15, 16).It is not known whether the properties that induce susceptibility to sPLA 2 in artificial membranes also contribute to the vulnerability of biological membranes to attack by the enzyme. In order to address this issue, we manipulated various properties of erythrocyte membranes by preparing different types of ghosts as explained in the accompanying particle (17). We found that the factors that determined the degree of susceptibility were increased exposure of phosphatidylserine, an anionic phospholipid, and increased membrane order. These interpretations agreed with those from studies of susceptibility using artificial membranes (5-16). The next question, then, is whether these same factors are important in the hydrolysis of intact cells by sPLA 2 under conditions at which they have become susceptible such as in the presence...

Normally, cell membranes resist hydrolysis by secretory phospholipase A 2 . However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A 2 . To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 °C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environmentsensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 °C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A 2 depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A 2 prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.