Intracellular pH and calcium in frog early distal tubule: effects of transport inhibitors.

1. The K+ channels of the apical membrane of the diluting segment (early distal tubule, EDT) of the frog are involved in the regulation of transepithelial NaCl transport. These channels are sensitive to pHi and intracellular Ca2+ (Ca2+i). Inhibition of transport by furosemide (frusemide) results in a compensatory increase in K+ channel activity. The aims of the present study were to determine whether pHi or Ca2+i were altered by furosemide, and to identify the means by which such changes were brought about. 2. Experiments were performed using single, microperfused EDT segments. Measurements of pHi and Ca2+i were made using the intracellular fluorescent probes, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and fura-2, respectively. 3. Furosemide increased pHi and Ca2+i. The intracellular alkalinization was the result of an alkaline shift in the set-point of the basolateral Na(+)-H+ exchanger. This response was dependent upon the increase in Ca2+i. 4. The increase in Ca2+i produced by furosemide was due to the release of Ca2+ from intracellular stores. Depletion of these stores, by 2,5-di-t-butylhydroquinone (TBQ) and caffeine, prevented the furosemide-induced changes in Ca2+ and pH. 5. Furosemide-induced activation of Na(+)-H+ exchange was prevented by the calmodulin antagonist, W-7. 6. Thus furosemide elicits a rise in Ca2+i which, via calmodulin, results in activation of Na(+)-H+ exchange. The resulting intracellular alkalinization would be expected to increase channel activity.