INTRACELLULAR PH AND CALCIUM IN FROG EARLY DISTAL TUBULE - EFFECTS OFTRANSPORT INHIBITORS

1. The K+ channels of the apical membrane of the diluting segment (ear ly distal tubule, EDT) of the frog are involved in the regulation of t ransepithelial NaCl transport. These channels are sensitive to pH(i) a nd intracellular Ca2+ (Ca-i(2+)). Inhibition of transport by furosemid e (frusemide) results in a compensatory increase in K+ channel activit y. The aims of the present study were to determine whether pH(i) or Ca -i(2+) were altered by furosemide, and to identify the means by which such changes were brought about. 2. Experiments were performed using s ingle, microperfused EDT segments. Measurements of pH(i) and Ca-i(2+) were made using the intracellular fluorescent probes, 2',7'-bis(carbox yethyl)5,6-carboxyfluorescein (BCECF) and fura-2, respectively. 3. Fur osemide increased pH(i) and Ca-i(2+). The intracellular alkalinization was the result of an alkaline shift in the set-point of the basolater al Na+-H+ exchanger. This response was dependent upon the increase in Ca-i(2+). 4. The increase in Ca-i(2+) produced by furosemide was due t o the release of Ca2+ from intracellular stores. Depletion of these st ores, by 2,5-di-t-butylhydroquinone (TBQ) and caffeine, prevented the furosemide-induced changes in Ca2+ and pH. 5. Furosemide-induced activ ation of Na+-H+ exchange was prevented by the calmodulin antagonist, W -7. 6. Thus furosemide elicits a rise in Ca-i(2+) which, via calmoduli n, results in activation of Na+-H+ exchange. The resulting intracellul ar alkalinization would be expected to increase channel activity.