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

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH⁺₄ transport on intracellular pH (pH_i) 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). pH_i was monitored using the fluorescent dye BCECF. In the absence of NH⁺₄, the mean pH_i was 7.16. Luminal addition of 20 mM NH⁺₄ caused a rapid intracellular acidification (dpH_i/dt = 11.1 U/min) and reduced the steady state pH_i to 6.67 (ΔpH_i = 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 pH_i 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 pH_i was 6.61 and net ammonium absorption was 12 pmol/min/ mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pH_i to increase abruptly (dpH_i/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 pH_i 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.