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.
Highlights
Under normal conditions, healthy cell membranes resist catalysis by secretory phospholipase A21 [1,2,3,4]
Effect of Ionomycin—As shown in Fig. 1, the extent of fatty acid release from erythrocyte membranes in the presence of secretory phospholipase A2 (sPLA2) was greatly enhanced by a 10-min prior incubation of the cells with ionomycin
Control experiments in which Ca2ϩ was replaced by EGTA in the extracellular medium revealed that this and the other results described below for ionomycin were due to Ca2ϩ entry into the cell rather than direct effects of the ionophore
Summary
Materials—Erythrocytes were obtained from healthy individuals undergoing routine physicals at Brigham Young University McDonald Health Center. Phospholipid Extraction and Thin Layer Chromatography—Washed erythrocytes (30 l) were suspended in MBSS to a final volume of 1 ml (about 1.5 ϫ 108 cells/ml) and incubated in the presence or absence of 0.3 M ionomycin with or without AppD49 sPLA2 for 20 min at 37 °C. Laurdan (2.5 M final) was added to samples of erythrocytes prepared as described above for fluorescence spectroscopy. Large comparisons of hydrolysis or light scattering data among many groups sharing some of the same blood samples Since multiple (two to three) treatment groups were compared with the same standard in these cases, a correction was made to the critical value of p accepted as indicating statistical significance (traditionally 0.05) according to the formula p ϭ 1– 0.951/n, where n ϭ the number of comparisons. For n ϭ 2, the critical value of p ϭ 0.025; for n ϭ 3, the critical value of p ϭ 0.017
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
