PLASMA-MEMBRANE NA+-H+ ANTIPORTER AND H+-ATPASE IN THE MEDULLARY THICK ASCENDING LIMB OF RAT-KIDNEY

To characterize H+ transport mechanisms in a fresh suspension of rat medullary thick ascending limb (MTAL) tubules, we have monitored intracellular pH (pH(i)) with use of the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. First, a Na+-H+ antiporter was identified in bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered media at 25-degrees-C. pH(i) recovery of Na-depleted acidified cells was dependent on extracellular sodium concentration, which was inhibited by amiloride in a manner consistent with simple competitive interaction with one external transport site (amiloride K(i) = 1.5-2.1 x 10(-5) M); Na-induced pH(i) recovery of acidified cells was electroneutral since it was not affected by 5 or 100 mM extracellular potassium in the presence or absence of valinomycin. Second, at 37-degrees-C, pH(i) recovery after acute intracellular acidification caused by 40 mM acetate addition to cell suspension was inhibited 36% by 200-400 nM bafilomycin A1, a macrolide antibiotic that specifically inhibits vacuolar-type H+-ATPase at submicromolar concentrations. In addition, amiloride-insensitive pH(i) recovery was inhibited by bafilomycin A1, 10(-3) M N-ethylmaleimide, and 10(-4) M preactivated omeprazole but not by 10(-5) M vanadate, 10(-4) M SCH 28080, or removal of extracellular potassium. Also, metabolic inhibition by absence of substrate, 10(-4) M KCN, or 5 x 10(-4) M iodoacetic acid inhibited amiloride-insensitive pH(i) recovery. The inhibitory effects of absence of metabolic substrate and iodoacetic acid were removed by reexpoure to glucose and L-leucine and by exogenous ATP, respectively. Finally, at 37-degrees-C, in bicarbonate-free HEPES-buffered medium, pH 7.4, resting pH(i) was 7.45 +/- 0.01 and was lowered to 7.40 +/- 0.01 (P < 0.05) by 2 x 10(-3) M amiloride and to 7.41 +/- 0.02 (P < 0.05) by 200-400 nM bafilomycin A1. We conclude that both Na+-H+ antiporter and plasma membrane H+-ATPase of the vacuolar type mediate proton extrusion from rat MTAL cells.