Ion regulation of phosphatidylserine and phosphatidylethanolamine outside-inside translocation in human erythrocytes.
In previous publications, we have shown, by using spin-labeled derivatives, that the translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer of human erythrocyte membrane is a protein-mediated phenomenon, which requires hydrolisable Mg2+-ATP. The inhibition by intracellular Ca2+ (0.2 microM) or by extracellularly added vanadate (50 microM) was reported (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755; Zachowski, A., Favre, E., Cribier, S., Hervé, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). The present article gives further insight into the effects of intracellular and extracellular ions on the aminophospholipid translocation in human erythrocytes. By measuring the cell ATP concentration, we now show that the inhibitory effect of intracellular calcium on spin-labeled aminophospholipid translocation is partly due to the ATP depletion, which follows the increased consumption by the calcium pump. However, a direct inhibitory effect of cytosolic Ca2+ on the aminophospholipid translocase can be demonstrated by measuring the initial rate of aminophospholipid translocation in the presence of variable amounts of intracellular calcium, at fixed ATP concentrations. Moreover, the transmembrane equilibrium distribution of phosphatidylserine and phosphatidylethanolamine are affected differently by Ca2+: when cytosolic Ca2+ concentration is increased, alteration of phosphatidylethanolamine distribution begins as soon as the inward translocation is affected by Ca2+ (approx. 50 nM), whereas phosphatidylserine distribution remains unchanged within a large inhibitory range of cytosolic Ca2+ concentrations and decreases above 0.2 microM of free Ca2+ within the cytosol. Decrease of the intracellular Mg2+ concentration below its physiological value (approx. 2 mM) results in the inhibition of aminophospholipid inward transport, whereas increase of Mg2+ concentration does not modify this transport. If Mn2+ is substituted for Mg2+, part of the aminophospholipid translocation is maintained, whereas if Co2+ is substituted for Mg2+, the rapid translocation is completely abolished. Concentrations as high as a millimolar of extracellular Ca2+, Mg2+ or Mn2+ have no effect on the aminophospholipid translocation. The less usual cations Cr3+, Fe2+, Cu2+, Sn2+ and Eu3+ are also uneffective. With extracellular Ni2+ or Co2+, some inhibition can be observed, half inhibition by Ni2+ corresponding to 500 microM. Vanadyl (VO2+), on the other hand, is a potent inhibitor of the aminophospholipid translocation when applied on the extracellular surface, half-inhibition being reached around 30 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000097:blood; D001692:Biological Transport; D002118:Calcium / Q000097:blood; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007477:Ions; D007700:Kinetics; D010714:Phosphatidylethanolamines / Q000097:blood*; D010718:Phosphatidylserines / Q000097:blood*; D014638:Vanadates / Q000494:pharmacology
D007477:Ions; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014638:Vanadates; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium