Shrinkage-induced activation of the Na+/H+ exchanger in Ehrlich ascites tumor cells: mechanisms involved in the activation and a role for the exchanger in cell volume regulation

Amiloride-sensitive, Na(+)-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na+ influx is also observed. This indicates that hypertonic challenge activates a Na+/H+ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pHi, but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mM HCO3(-), but not in nominally HCO3(-)-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.