Abstract
To characterize NH4+ transport in the renal medullary thick ascending limb (MTAL), cell pH was monitored with the use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in suspensions of rat MTAL tubules in CO2-free media. Abrupt exposure to NH4Cl led to initial cell alkalinization (NH3 entry) followed by cell acidification due to NH4+ influx. The latter had 1 microM amiloride- and barium-sensitive components; the barium effect was unchanged when K+ conductances were completely blocked by quinidine, as assessed with the use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine. NH4+ entry-induced depolarization was abolished by 1 microM amiloride, but it was unaffected by barium. In NH(4+)-free media, barium, verapamil, and raising of the extracellular K+ concentration alkalinized MTAL cells; imposing an outward directed K+ gradient in Na(+)-free medium induced cellular acidification, which was abolished by barium and verapamil but not by other K+ channel and Na+/H+ antiport inhibitors (quinidine and 2 mM amiloride). As measured with a K(+)-selective extracellular electrode, a component of K+ efflux (in presence of furosemide, ouabain, and quinidine) was stimulated by decreasing the extracellular pH from 7.4 to 7.0 and inhibited by barium and verapamil. It was also demonstrated that the K+/H+ antiporter transports NH4+ better than H+ at physiological NH4+ and H+ concentrations. These results demonstrate the presence in MTAL cells of two novel NH4+ transport pathways, amiloride-sensitive NH4+ conductance and barium- and verapamil-sensitive K+/NH4+(H+) antiport.
Highlights
From the Laboratoire de Physiologieet Endocrinologie Cellulaire Rknale, INSERM Unite‘356, Uniuersite Pierre et Marie Curie and Hhpital Broussais, Paris, France
7.25 l.I.I.III.l.I.I.l any indirectly activated H+-OH- conductanceas being responsible for this K+ efflux-induced intracellular acidification. They confirm that K+/H+ exchangeis electroneutral since the K+efflux-induced cell acidification occurredwithout change in thecell membrane PD in thepresence of quinidine or amiloride.the H'KATPase-specific inhibitor SCH 28080 (24) had no effect ( n = 4, data not shown).Of interest is the fact that 0.1 mM verapamil, used initially as another K+channel blocker (111,abolished the K+efflux-induced intracellular acidification (Fig.[8] A )
0.1 mM verapamil alkalinized, within a few seconds, MTAL cells incubated in a NH;-free Hepesms medium by 0.06 0.01 pH unit ( p < 0.01)without interfering with Na+/H+antiport activity studied as described above(data not shown).alkalinizing effects of barium and verapamil were still observed inthe absence of extracellular calcium (Hepeas-buffered medium nominallyCa2+-freeplus 5 x M EGTA), which excludesany indirect effect secondary to possible inhibition of Ca" channels by theseagents(data not shown)
Summary
From the Laboratoire de Physiologieet Endocrinologie Cellulaire Rknale, INSERM Unite‘356, Uniuersite Pierre et Marie Curie and Hhpital Broussais, Paris, France. NH; absorption by the MTAL is to influx.The latter had[1] p~amiloride- and barium-a key step in the renal tubular handlingof ammonia, all the sensitive components;the barium effect was unchanged more so since it is subjected to regulation. In NH;-free media, barium, verapamil, anradising of the extracellular K+ concentrationalkalinized abolic acidosis is associated with an adaptive increase iMn TAL ammonia absorption (3);the mechanism(so)f this adaptation is unknown, . The knowledge of the NH; transport pathways which was abolished by barium and verapamil but noptresent in MTAL cells is a prerequisite for understanding the byother K+ channelandNa+/H+antiportinhibitors mechanisms of regulation of MTAZ, NH; absorption. (quinidine and[2] m~ amiloride).As measured with aK+- Studies using plasma membranvesicles prepared from rabselective extracellular electrode, a componentof K+ ef- bit MTAL cells have shown thaNt H; may be accepted by the K’
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