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 alkal inization is observed after hypertonic challenge in Ehrlich ascites tu mor 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+/Hexchanger. Activation occurs after several types of hypertonic challen ge, is a graded function of the osmotic challenge, and is temperature- dependent. Observations on single cells reveal a considerable variatio n in the shrinkage-induced changes in cellular pH(i), but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is stro ngly reduced in ATP-depleted cells. Furthermore, it is inhibited by ch elerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast , Calyculin A, an inhibitor of protein phosphatases PPI and PP2A, stim ulates shrinkage-induced alkalinization. Osmotic activation of the exc hanger is unaffected by removal of calcium from the experimental mediu m, and by buffering of intracellular free calcium with BAPTA. At 25 mM HCO3-, but not in nominally HCO3--free medium, Na+/H+ exchange contri butes significantly to regulatory volume increase in Ehrlich cells. Un der isotonic conditions, the Na+/H+ exchanger is activated by ionomyci n, an effect which may be secondary to ionomycin-induced cell shrinkag e.