The inhibitory effect of cytoplasmic Ca on Na-pump-mediated Na-K exchange was investigated in intact red cells under conditions of constant cell volume, membrane potential and inorganic ion composition. The ionized cytoplasmic Ca concentration ( [Ca2+]i) was controlled using the ionophore A23187. In normal cells, ouabain-sensitive 24Na efflux was inhibited with an apparent affinity for [Ca2+]i which depended on the concentration of A23187; 50% inhibition required 20-40 microM and 160-300 microM-cytoplasmic Ca2+ with 10 microM and 0.63 microM-A23187 respectively. Cytoplasmic Ca also affected cell ATP content which fell rapidly on addition of A23187 and subsequently increased, steadied or continued to fall more slowly depending on the Ca and A23187 concentrations. Half-maximal fall required 5-15 microM and 110-170 microM-cytoplasmic Ca2+ at 10 microM and 0.63 microM-A23187 respectively. Removal of Ca from the cells failed to reverse either the Na pump inhibition or the fall in cell ATP. In ATP-enriched cells cytoplasmic Ca caused inhibition of ouabain-sensitive 24Na efflux in an A23187-dependent manner with apparent affinities for [Ca2+]i similar to those observed in the normal cells. Inhibition was complete at high [Ca2+]i. As in the normal cells, the ATP content of the cells fell in the presence of cytoplasmic Ca, but always remained above 1.2 m-mole/l. cells. This was higher than the ATP content of Ca-free normal intact cells. A23187 had no effect on the inhibition by Ca of ouabain-sensitive ATPase activity in isolated red cell membrane preparations. Both under conditions near optimal for Na-K-ATPase activity and under conditions resembling those in the cytoplasm, inhibition was half-maximal at about 25 microM-Ca2+ and in the latter case complete at below 400 microM-Ca2+. The apparent ATP-dependence of ouabain-sensitive Na efflux in the presence of cytoplasmic Ca was distinctly different in the normal and ATP-enriched cells but in both groups of cells it was similar for data obtained with high and low concentrations of A23187. The data for Na pump inhibition by cytoplasmic Ca in the intact cells were well fitted by several kinetic models involving either [Ca2+]i or CaATP as the inhibitory species and a low affinity dependence of pump activity on MgATP or total ATP. However, for any model, the apparent affinities for CaATP or for Ca2+ required to fit the ATPase data were 2.5-10 times higher than those required to fit the data for Na efflux.(ABSTRACT TRUNCATED AT 400 WORDS)
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