Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes.
Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.
de Jong
K
K
Children's Hospital Oakland Research Institute, California 94609, USA.
Geldwerth
D
D
Kuypers
F A
FA
D000814:Aniline Compounds; D017304:Annexin A5; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood / Q000494:pharmacology; D004907:Erythrocyte Deformability; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008563:Membrane Lipids / Q000097:blood*; D018384:Oxidative Stress; D010659:Phenylhydrazines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000097:blood; D010743:Phospholipids / Q000097:blood*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism; D014966:Xanthenes
D000814:Aniline Compounds; D017304:Annexin A5; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D010659:Phenylhydrazines; D010718:Phosphatidylserines; D010743:Phospholipids; D013439:Sulfhydryl Reagents; D014966:Xanthenes; C030299:phenylhydrazine; C059715:Fluo-3; D000001:Calcimycin; D013925:Thromboplastin; D006861:Hydrogen Peroxide; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide; D002118:Calcium