Effects of ammonium on intracellular pH in rat medullary thick ascending limb: Mechanisms of apical membrane NH+4 transport

Abstract

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH+4 transport on intracellular pH (pHi) and the mechanisms of apical membrane NH+4 transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO-3-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH+4, the mean pHi was 7.16. Luminal addition of 20 mM NH+4 caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (ΔpHi = 0.5 U), indicating that apical NH+4 entry was more rapid than entry of NH3. Luminal furosemide (10-4 M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH+4 entry occurred via both furosemide (Na+-NH+4-2Cl- cotransport) and bariumsensitive pathways. The role of these pathways in NH+4 absorption was assessed under symmetric ammonium conditions. With 4 mM NH+4 in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/ mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH+4. In symmetric 4 mM NH+4 solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH+4 absorption markedly acidifying the cells and maneuvers that inhibit apical NH+4 uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na+-NH+4-2Cl- cotransport; (c) NH+4 also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.