Cl (-) -dependent NH4+ transport mechanisms in medullary thick ascending limb cells
Amlal, H.; Paillard, M.; Bichara, M.
American Journal of Physiology 267(6 Pt 1): C1607-C1615
1994
ISSN/ISBN: 0002-9513 PMID: 7810602 Document Number: 424933
To characterize Cl-dependent NH-4+ 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 CO-2-free media. Exposure of MTAL cells to 4 mM NH-4Br caused, after an initial cell alkalinization due to NH-3 entry, an NH-4+-induced fall in pH-i that was apprx 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 NH-4+ 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 apprx 0.2 pH units in the presence of 4 mM NH-4Br, which thus indicated nonelectrogenic (NMDG-Cl)-dependent NH-4+ 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 NH-4+-induced fall in pH-i was reduced apprx 33% by 10 mM Ba+, apprx 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+/NH-4+ 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+(NH-4+)-2Cl- cotransport blockade) had no effect on pH-i in the absence of ammonia despite marked hyperpolarization, but rapidly alkalinized the cells by apprx 0.08 pH units in the presence of 4 mM NH-4+-SCN, which thus indicated nonelectrogenic Cl--dependent NH-4+ 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+(NH-4+)-2Cl- cotransport, a Ba+- and furosemide-sensitive NH-4+(K+)-Cl- cotransport takes place in MTAL cells.