CL--DEPENDENT NH4+ TRANSPORT MECHANISMS IN MEDULLARY THICK ASCENDING LIMB CELLS

To characterize Cl--dependent NH4+ transport mechanisms in renal medullary thick ascending limb (MTAL), intracellular pH (pH(i)) and membrane potential (PD) were monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and 3,3'-dipropylthiadicarbocyanine, respectively, in suspensions of rat MTAL tubules in CO2-free media. Exposure of MTAL cells to 4 mM NH4Br caused, after an initial cell alkalinization due to NH3 entry, an NH4+-induced fall in pH(i) that was similar to 67% less pronounced in Cl--free than in Cl--containing media. The following experiments were performed in the presence of 1 mu M amiloride to block the MTAL NH4+ conductance. When cells were preincubated in a Cl--free gluconate medium in which K+ and Cl- conductances are greatly reduced, abrupt addition of 100 mM N-methyl-D-glucamine (NMDG)-Cl had no effect on cell PD and pH(i) in the absence of ammonia, but acutely acidified the cells by similar to 0.2 pH units in the presence of 4 mM NH4Br, which thus indicated nonelectrogenic (NMDG-Cl)-dependent NH4+ influx. The latter also occurred in a Cl--free thiocyanate medium in which the Cl- conductance was blocked by 0.1 mM diphenylamine-2-carboxylate (DPC). An NMDG-Cl-dependent NH4+-induced fall in pH(i) was reduced similar to 33% by 10 mM Ba+, similar to 84% by 0.1 mM bumetanide, and 100% by 1.5 mM furosemide, whereas 1 mM hydrochlorothiazide had no effect; inhibition by Ba+ was observed even in the presence of 0.1 mM verapamil added to block both K+ channels and K+/NH4+ antiport. Conversely, diluting Cl--loaded cells into a Cl--free gluconate medium in the presence of 0.1 mM DPC and 0.1 mM bumetanide [Na+-K+(NH4+)-2Cl(-) cotransport blockade] had no effect on pH(i) in the absence of ammonia despite marked hyperpolarization, but rapidly alkalinized the cells by similar to 0.08 pH units in the presence of 4 mM NH4+-SCN, which thus indicated nonelectrogenic Cl--dependent NH4+ outflux. The latter was also observed in Na+-free media, and was abolished by 10 mM Ba+ and 1.5 mM furosemide. We conclude that, in addition to bumetanide(furosemide)-sensitive Na+-K+ (NH4+)-2Cl(-) cotransport, a Ba+- and furosemide-sensitive NH4+ (K+)-Cl- cotransport takes place in MTAL cells.