Na+-H+ exchanger in proximal cells isolated from rabbit kidney. I. Functional characteristics

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.