Volume regulation by Amphiuma red blood cells: cytosolic free Ca and alkali metal-H exchange

Abstract

Osmotic swelling of Amphiuma red blood cells results in activation of electroneutral K-H exchange, whereas cell shrinkage activates an electroneutral Na-H exchange. These K-H and Na-H exchangers function to restore cell volume to normal after cell swelling and shrinkage, respectively. Our previous studies have suggested that Ca plays a role in volume-dependent activation of K-H exchange. In the present studies, intracellular free Ca levels were measured employing the Ca-sensitive extracellular dye arsenazo III and a previously described null-point method. Control values for intracellular free Ca averaged 0.46 +/- 0.15 microM. Cell shrinkage caused this value to decrease to 0.16 +/- 0.11 microM, whereas either cell swelling or addition of 5 microM A23187 resulted in saturation of intracellular Ca buffers, suggesting that both treatments caused an increase in intracellular free Ca. In the presence of 7 microM A23187, the rate of K-H exchange displayed a hyperbolic relationship as a function of extracellular Ca (Cao). The apparent half-maximal concentration for Cao (in the presence of 7 microM A23187) was 0.27 mM for osmotically swollen cells and 1.9 mM for cells in isotonic medium, suggesting that the Ca affinity of a modulating site is increased in swollen cells. Inhibitors of Ca-mediated processes, such as quinidine and the phenothiazines, inhibited K-H exchange. In contrast, the phenothiazines chlorpromazine and trifluoperazine stimulated Na-H exchange by osmotically shrunken cells. These results suggest that increases in intracellular free Ca are involved in stimulating K-H exchange while repressing Na-H exchange in Amphiuma red blood cells.