Sodium-proton exchange in human ileal brush-border membrane vesicles

Abstract

This study examines the characteristics of Na+ and H+ transport as well as Na+H+ exchange in human ileal brush-border membrane vesicles from organ donor intestine. 22Na+ uptake into vesicles and the fluorescence quenching of Acridine orange were employed to measure Na+ and H+ transport, respectively. Concentrative uptake of 22Na+ (4-fold overshoot above equilibrium) was observed under conditions of an outward proton gradient (pHi 5.5; pHo 7.5). Voltage-clamping (Ki+ = Ko+ + valinomycin) reduced the uptake of 22Na+ by 40–50% indicating the presence of Na+ conductance. Dissipation of the Acridine orange fluoresence quench in ileal vesicles with a performed pH gradient (pHi 5.5; pHo 7.5) was accelerated by either external Na+ or voltage-clamping in the absence of Na+. The effects of Na+ and voltage-clamping were additive under the above conditions. In the absence of a pH gradient, Acridine orange quenching was induced by intravesicular Na+ as well as an interior negative K+ diffusion potential. In voltage-clamped BBMV, pH-driven Na+ uptake was inhibited by amiloride (Ki = 140 μM). The initial rate of pH-driven Na+ uptake was saturable and conformed to Michaelis-Menten kinetics with apparent Km and Vmax values of 27 ± 1 mM and 47 ± 1 nmol·(mg protein)−1·(3 s)−1, respectively. Li+ and NH4+, but not Cs+, K+, Rb+ or choline+ inhibited pH gradient-driven 22Na+ uptake. The results demonstrate in human ileal brush-border membrane vesicles the presence of an Na+/H+ exchanger and conductive transport pathways for Na+ and H+.