Na+ transport in human proximal colonic apical membrane vesicles

Abstract

The mechanisms of Na+ movement across colonocyte plasma membranes in the human colon are not well understood. Current studies were undertaken to investigate Na+ transport pathways in apical membranes of proximal organ donor colons. Purified apical membrane vesicles and rapid filtration 22Na-uptake techniques were used. An outwardly directed H(+)-gradient (pH 5.5 in/7.5 out) increased 22Na uptake into these vesicles. H+ gradient-driven 22Na uptake was significantly reduced by voltage clamping with K+/valinomycin, but was significantly stimulated by creation of an inside-negative potential. Potential sensitive 22Na uptake was inhibited by Na+ channel inhibitors phenamil and benzamil. Electroneutral 22Na uptake was insensitive to phenamil and benzamil, but was inhibited by amiloride, 5-(N,N-dimethyl)amiloride, 5-(N,N-hexamethylene)amiloride, and 5-(N-ethyl-N-isopropyl)amiloride. Electroneutral 22Na uptake showed saturation kinetics with an apparent Michaelis constant for Na+ of 11.8 +/- 2.4 mmol/L and a maximal velocity of 2.5 +/- 0.6 nmol.mg protein-1 x 5 s-1. The mechanism of amiloride inhibition was noncompetitive with an inhibitor constant for amiloride of 325 mumol/L. Acetazolamide, furosemide, bumetanide, 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stibene, and 4,4'-di-isothiocyanatostilbene-2,2'-disulfonic acid (1 mmol/L each) failed to inhibit 22Na uptake. Li+ and NH4+ (but not Cs+, K+, or choline+) inhibited H(+)-gradient driven 22Na uptake. Na+ transport in human proximal colonic apical membrane vesicles involves both conductive Na+ transport and an electroneutral Na(+)-H+ exchange.