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J. Biol. Chem., Vol. 276, Issue 25, 22732-22741, June 22, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/25/22732    most recent M010880200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Smith, S. K. Articles by Bell, J. D. Search for Related Content PubMed PubMed Citation Articles by Smith, S. K. Articles by Bell, J. D. Social Bookmarking                      What's this?

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

Samantha K. Smith, Amelia R. Farnbach, Faith M. Harris, Andrea C. Hawes, Laurie R. Jackson, Allan M. Judd, Rebekah S. Vest, Susana Sanchez^§, and John D. Bell^¶

From the  Department of Zoology, Brigham Young University, Provo, Utah 84602 and the ^§ Laboratory for Fluorescence Dynamics, University of Illinois at Champaign-Urbana, Urbana, Illinois 61801

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A₂ (sPLA₂). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA₂. Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A₂ 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 sPLA₂ is a consequence of increased order of membrane lipids.

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