Mechanisms by Which Intracellular Calcium Induces Susceptibility to Secretory Phospholipase A2 in Human Erythrocytes

Abstract

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...

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A2

“…After initiating data acquisition, ADIFAB was added followed 100 s later by ionomycin (300 nM) or DMSO, and 700 s later by sPLA 2 (1 µg/mL). The results were quantified by calculation of the generalized polarization (GP) and fit to a double exponential equation by nonlinear regression as described (12). The amount of hydrolysis at 100 s following sPLA 2 addition was then calculated using parameter values from the regression results.…”

“…GP was calculated and the data fit by nonlinear regression as described in Experimental Procedures. The values shown in the figure were calculated from the fits for the 1000 s time point to represent values after stability was reached (12). The curve shown in part B was obtained by a fit of the data points to an arbitrary function and has no theoretical basis.…”

“…Stock solutions were suspended at a concentration of 0.1 mg/mL in 50 mM KCL and 3 mM NaN 3 , and stored at 4°C. Prior studies have demonstrated that this isozyme reports qualitatively the susceptibility of cells to human groups IIa and V sPLA 2 (12,18).…”

“…Human erythrocytes are amenable to such investigations because of their simplicity. In previous studies, it was observed that erythrocytes normally resist the action of sPLA 2 , but that upon the addition of a calcium ionophore such as ionomycin, they become susceptible (12). This increase in susceptibility appears to be linked to an accompanying increase in the order of membrane lipids (12,13).…”

“…In previous studies, it was observed that erythrocytes normally resist the action of sPLA 2 , but that upon the addition of a calcium ionophore such as ionomycin, they become susceptible (12). This increase in susceptibility appears to be linked to an accompanying increase in the order of membrane lipids (12,13). Nevertheless, one question that remained unresolved was whether the critical issue for increased susceptibility was the average level of membrane order or the spatial distribution of membrane regions of differential order.…”