Inhibition of the calcium pump by high cytosolic Ca2+ in intact human red blood cells.
1. The inhibitory effect of high intracellular calcium on the saturated Ca2+ efflux through the Ca2+ pump (Vmax) was investigated in intact human red cells. Cells were loaded with Ca2+ by exposure to the calcium ionophore A23187, at different external Ca2+ concentrations ([Ca2+]o). Ca2+ extrusion by the pump was followed after either ionophore removal or Co2+ addition. 2. fifty per cent inhibition of Vmax was obtained with total intracellular calcium ([CaT]i) of approximately 3 mmol/l cells. For any given initial Ca2+ load, Vmax showed no tendency to increase as [CaT]i was progressively reduced during Ca2+ efflux. This suggests that the pump Vmax was determined by the magnitude of the initial [Ca2+]i. 3. To estimate [Ca2+]i from [CaT]i in Co(2+)-loaded cells, the possible competition between Co2+ and Ca2+ for the known cytoplasmic Ca2+ buffers (alpha-buffers) was investigated first. Comparison between Ca2+ efflux after either Co2+ exposure or ionophore wash-out showed that the efflux patterns were essentially identical, down to the lowest measurable [CaT]i. This indicates that Co2+ does not compete with Ca2+ for the alpha-buffers. Hence, since [Ca2+]i = alpha [CaT]i, and alpha approximately 0.15-0.35, the initial [Ca2+]i load for 50% Vmax inhibition was between 0.4 and 1.1 mM. 4. Ancillary new findings demonstrated that, unlike the situation with alpha-buffers, Co2+ displaced Ca2+ from the cell-incorporated calcium chelator benz-2, and that benz-2 incorporation had no effect on Co(2+)-exposed Ca2+ pump desaturation. This validates the use of benz-2 to study Ca2+ pump kinetics in intact cells.
Physiological Laboratory, University of Cambridge.
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Pereira
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Samellas
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Tiffert
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Lew
D001667:Binding, Competitive; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009930:Organic Chemicals
C041685:Benz2; D002614:Chelating Agents; D009930:Organic Chemicals; D003035:Cobalt; D000252:Calcium-Transporting ATPases; D002118:Calcium