Intracellular pH Regulation in the $3 Segment of the Rabbit Proximal Tubule in HCO j-free Solutions

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused $3 segment of the rabbit proximal tubule. Experiments were conducted in HCO~-free solutions, pH~ recovered from an acid load imposed by an NH~ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na + from bath and lumen caused pHi to decrease by ~0.6, whereas Na + readdition caused complete pH~ 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 pH~ recovery produced by the readdition of Na + to the bath and lumen was about nlnefold 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 -30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited ~66% of the phi recovery induced by the readdition of 29 mM Na + to the lumen only. The removal of external Acreduced the pHi recovery rate from an NH~'-induced acid load by -47%, and that elicited by Na § readdition, by ~67%. Finally, when bilateral removal of Na + was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi ~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 ~57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO~, three transport systems contribute to acid extrusion by $3 cells: (a) a Na +independent mechanism, possibly an H + pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Acand luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na +, was ~ 13, ~30, and ~57%, respectively. I N T R O D U C T I O N The mammalian proximal tubule is a major site of transepithelial acid secretion. This process, which results in the acidification of the luminal fluid and the net reabAddress reprint requests to Dr. Walter F. Boron, Department o f Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06510-8026. J. GIgN. PHYSIOL. ~ The Rockefeller University Press 9 0022-1295/88/09/0369/25 $2.00 369 Volume 92 September 1988 369-393 on Jne 1, 2017 D ow nladed fom Published September 1, 1988