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Determination of NH 4 (cid:49) /NH 3 ﬂuxes across apical membrane of macula densa cells: a quantitative analysis

Laamarti, and Jean-Yves Lapointe. Determination of NH 4 (cid:49) /NH 3 ﬂuxes across apical membrane of macula densa cells: a quantitative analysis. Am. J. Physiol. 273 ( Renal Physiol. 42): F817–F824, 1997.—Luminal addition of 20 mM NH 4 (cid:49) produced a rapid acidiﬁcation of rabbit macula densa (MD) cells from 7.50 (cid:54) 0.06 to 6.91 (cid:54) 0.05 at an initial rate of 0.071 (cid:54) 0.008 pH unit/s. In the luminal presence of 5 µM bumetanide, 5 mM Ba 2 (cid:49) or both, the acidiﬁcation rate was reduced by 57%, 35% and 93% of control levels. In contrast, intracellular pH (pH i ) recovery after removing luminal NH 4 (cid:49) was unaffected by bumetanide and Ba 2 (cid:49) but was sensitive to 1 mM luminal amiloride (71% inhibition). The bumetanide-sensitive acidiﬁcation rate represents most certainly the NH 4 (cid:49) ﬂux mediated by the apical Na (cid:49) :K (cid:49) (NH 4 (cid:49) ):2Cl (cid:50) cotransporter, but the Ba 2 (cid:49) -sensitive portion does not seem to be associated with the apical K (cid:49) channels previously characterized by us. The effects of NH 4 (cid:49) entry across the apical membrane were simulated using a simple model involving ﬁve adjustable parameters: apical and basolateral permeabilities for NH 4 (cid:49) and NH 3 and a parameter describing a pH-regulating mechanism. The model shows that the apical membrane of MD cells is much more perme-able to NH 3 than it is to NH 4 (cid:49) and, under control conditions, the apical NH 4 (cid:49) ﬂux appears surprisingly high (11–20 mM/s) and challenges the notion that MD cells present a low intensity of ionic transport. 7.2, 7.6, and 8.0. Calibration curves were obtained, and one can see that the ﬂuorescence ratios converge to the same value in acidic conditions (6.4–6.8), whereas all the ratios were distrib-uted within (cid:54) 7% of the mean at a pH i of 7.6. As a complete calibration could not be performed in each experiment, one or two calibration points (including pH i (cid:53) 7.6) were obtained at the end of each experiment, and the calibration curve which best satisﬁed these points was used to scale the measured ﬂuorescence ratios. Statistics . Statistical signiﬁcance of a difference between two average results was tested using Student’s t -test for paired sample. P (cid:44) 0.05 was considered signiﬁcant.