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Physiological Laboratory, University of Cambridge.

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.

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