Determination of NH 4 + /NH3fluxes across apical membrane of macula densa cells: a quantitative analysis

Luminal addition of 20 mM NH4+ produced a rapid acidification of rabbit macula densa (MD) cells from 7.50 +/- 0.06 to 6.91 +/- 0.05 at an initial rate of 0.071 +/- 0.008 pH unit/s. In the luminal presence of 5 microM bumetanide, 5 mM Ba2+ or both, the acidification rate was reduced by 57%, 35% and 93% of control levels. In contrast, intracellular pH (pHi) recovery after removing luminal NH4+ was unaffected by bumetanide and Ba2+ but was sensitive to 1 mM luminal amiloride (71% inhibition). The bumetanide-sensitive acidification rate represents most certainly the NH4+ flux mediated by the apical Na+:K+ (NH4+):2Cl- cotransporter, but the Ba(2+)-sensitive portion does not seem to be associated with the apical K+ channels previously characterized by us. The effects of NH4+ entry across the apical membrane were simulated using a simple model involving five adjustable parameters: apical and basolateral permeabilities for NH4+ and NH3 and a parameter describing a pH-regulating mechanism. The model shows that the apical membrane of MD cells is much more permeable to NH3 than it is to NH4+ and, under control conditions, the apical NH4+ flux appears surprisingly high (11-20 mM/s) and challenges the notion that MD cells present a low intensity of ionic transport.