Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

Abstract

We used the absorbance spectrum of 4'',5''-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3--free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by .apprx. 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produce by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only .apprx. 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited .apprx. 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by .apprx. 47%, and that elicited by Na+ readdition, by .apprx. 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial aciification, slowly at first, and then more rapidly, eventually reaching at pHi .apprx. 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N''N''-dicyclohexylcarbodiimide (10-4 M) to the lumen, but it was reduced .apprx. 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was .apprx. 13, .apprx. 30, and .apprx. 57%, respectively.