AJP - Renal Physiology, Vol 253, Issue 5 935-F944, Copyright © 1987 by American Physiological Society
Na+-H+ exchanger in proximal cells isolated from rabbit kidney. I. Functional characteristics
M. Bidet, M. Tauc, J. Merot, A. Vandewalle and P. Poujeol
Institut National de la Sante et de la Recherche Medicale, Unite 246, Centre d'Etudes Nucleaires de Saclay, Gif-Sur-Yvette, France.
The purpose of this study was to investigate the characteristics of the
Na+-H+ exchange in isolated proximal cells from rabbit kidney cortex. The
cells were prepared by mechanical dissociation and sequential passages
through nylon meshes. The intracellular pH (pHi) was measured in a
bicarbonate-free medium [extracellular pH (pHe) = 7.30], using the
fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF).
Resting pHi was 7.13 +/- 0.04 (n = 11) at 20-22 degrees C. Cells were acid
loaded with nigericin in choline solution and H+ efflux, induced by
extracellular Na+ (Nae), was calculated using a buffering power of 23.6 +/-
0.6 mmol.1-1.pH unit-1 (n = 19) estimated by NH4Cl exposure. In isolated
proximal cells, the Na+-H+ antiporter had an apparent Km for Nae of 86.7
+/- 1.5 mM (n = 4) and was competitively inhibited by amiloride with a Ki
of 33.3 +/- 6.4 X 10(-6) M (n = 3). Lowering pHe, inhibited the Na+-H+
exchanger. This inhibition was not purely competitive and the Ki was 40.4
+/- 12.7 nM (n = 3). The Na+-H+ exchange was greatly activated when the
cytoplasm was acidified. The intracellular H+ concentration dependence did
not follow simple Michaelis-Menten kinetics. Of the different cations
tested on pHi recovery, such as Li+, choline+, K+, and tetramethylammonium,
only Li+ induced an alkalinization of acidified cells similar to that of
Na+. 22Na influx measurements indicated that cellular depletion of Na+
stimulated Na+-H+ exchange. The results permit the conclusion that the
isolation procedures did not impair the main features of the Na+-H+
antiporter, at least as compared with those previously described in renal
brush-border membrane vesicles or in other cellular systems. The integrity
of the transporter in isolated proximal cells would permit the direct study
of its hormonal and metabolic control.
