Evidence against a proton pump in rabbit proximal convoluted tubule

H+/OH- transport in the absence of bicarbonate was studied in the rabbit proximal convoluted tubule (PCT) perfused in vitro using measurements of membrane potential and intracellular pH (pHi). Blockade of apical Na/H exchange led to a cell acidification of 0.64 +/- 0.1 pH units from a control pHi of 7.27 +/- 0.04. A bafilomycin-insensitive recovery of pHi of 0.05 +/- 0.02 pH units occurred, but pHi did not exceed electrochemical equilibrium. A larger, sustained acidification of 0.87 +/- 0.07 from an initial control pHi of 7.25 +/- 0.05 induced by bilateral Na removal left pHi substantially below electrochemical equilibrium. These results suggest the absence of Na-independent active proton extrusion. We also examined the possibility that a passive electrogenic proton leak may exist. The removal of luminal glucose and alanine led to a basolateral membrane hyperpolarization of 31.3 +/- 0.5 mV, which was associated with a cell acidification of 0.15 +/- 0.02 pH units. These responses were reversed by addition of luminal glucose and alanine but not by depolarization by basolateral barium, suggesting that luminal glucose and alanine effects on pHi were due to changes other than cell potential. We conclude that, in the absence of bicarbonate, all active proton extrusion in the rabbit PCT is dependent on active Na transport and that a proton leak is negligible.