title: Effect of intracellular magnesium on calcium extrusion by the plasma membrane calcium pump of intact human red cells.
abstract: 1. The effect of varying the concentration of intracellular magnesium on the Ca(2+)-saturated Ca(2+)-extrusion rate through the Ca2+ pump (phi max) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+]i dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [45Ca]CaCl2 equilibration, prior to measuring phi max at constant internal [MgT]i by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+]o. To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading-extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading-extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+]i. Ionophore removal was therefore used to estimate the [Mg2+]i dependence of the pump at levels below [MgT]i (approximately 2 mmol (340 g Hb)-1), whereas both ionophore removal and Co2+ block were used for higher [MgT]i levels. 4. [Mg2+]i was computed from measured [MgT]i using known cytoplasmic Mg(2+)-buffering data. The phi max of the Ca2+ pump increased hyperbolically with [Mg2+]i. The Michaelis parameter (K 1/2) of activation was 0.12 +/- 0.04 mmol (1 cell water)-1 (mean +/- S.E.M.). Increasing [MgT]i and [Mg2+]i to 9 mmol (340 g Hb)-1 and 2.6 mmol (1 cell water)-1, respectively, failed to cause significant inhibition of the phi max of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+]i, from 0.3 mmol (1 cell water)-1 in the oxygenated state to 1.2 mmol (1 cell water)-1 in the deoxygenated state, the Ca(2+)-saturated Ca2+ pump remains unaffected by [Mg2+]i at normal ATP levels.
authors.affiliation: Physiological Laboratory, University of Cambridge, UK.
authors.forename: J E
authors.initials: JE
authors.lastname: Raftos
authors.forename: V L
authors.initials: VL
authors.lastname: Lew
mesh_terms: D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D000001:Calcimycin; D008274:Magnesium; D002118:Calcium