Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.

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1. The effects of deoxygenation on cytoplasmic Ca2+ buffering, saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax), passive Ca2+ influx and physiological [Ca2+]i level were investigated in human red cells to assess whether or not their Ca2+ metabolism might be altered by deoxygenation in capillaries and venous circulation. 2. The study was performed in fresh human red cells maintained in a tonometer either fully oxygenated or deoxygenated. Cytoplasmic Ca2+ buffering was estimated from the equilibrium distribution of 45Ca2+ induced by the divalent cation ionophore A23187 and the Vmax of the Ca2+ pump was measured either by the Co(2+)-exposure method or following ionophore wash-out. The passive Ca2+ influx and physiological [Ca2+]i were determined in cells preloaded with the Ca2+ chelator benz-2 and resuspended in autologous plasma. 3. Deoxygenation increased the fraction of ionized Ca2+ in cell water by 34-74% and reduced the Vmax of the Ca2+ pump by 18-32%. 4. To elucidate whether or not these effects were secondary to deoxygenation-induced pH shifts, the effects of deoxygenation on cell and medium pH, and of pH on cytoplasmic Ca2+ binding and Ca2+ pump Vmax in oxygenated cells were examined in detail. 5. Deoxygenation generated large alkaline pH shifts that could be explained if the apparent isoelectric point (pI) of haemoglobin increased by 0.2-0.4 pH units in intact cells, consistently higher than the value of 0.15 reported for pure haemoglobin solutions. 6. In oxygenated cells, the fraction of ionized cell calcium, alpha, was little affected by pH within the 7.0-7.7 range. Ca2+ pump Vmax was maximal at a medium pH of about 7.55. Comparison between pH effects elicited by HCl-NaOH additions and by replacing Cl- with gluconate suggested that Vmax was inhibited by both internal acidification and external alkalinization. Since deoxygenation alkalinized cells and medium within a range stimulatory for Vmax, the inhibition observed was not due to pH. 7. There was no significant effect of deoxygenation on passive Ca2+ uptake, or steady-state physiological [Ca2+]i level. 8. The deoxygenation-induced reduction in Ca2+ binding capacity may result from the increased protonation of haemoglobin on deoxygenation and from binding of 2,3-diphosphoglyceric acid (2,3-DPG) and ATP to deoxyhaemoglobin; inhibition of the Ca2+ pump may result from shifts in the [Mg2+]i/[ATP]i ratio away from a near optimal stimulatory value in the oxygenated state.

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