Mechanisms of Na + transport in human distal colonic apical membrane vesicles

Abstract

Apical membrane vesicles purified from mucosal scrapings obtained from distal segments of organ donor colons and a 22Na-uptake technique were used to characterize the mechanism(s) of Na + transport into these vesicles. An outwardly directed H + gradient (pH 5.5 in/7.5 out) markedly increased uptake of 22Na into these vesicles. Osmolarity studies demonstrated that 22Na was taken up into the intravesicular space with minimal binding observed to the surface of the vesicles. Voltage clamping in the presence of K +/valinomycin reduced the H + gradient-dependent 22Na uptake into these vesicles by ≈ 45% and generation of an inside negative membrane potential significantly increased 22Na uptake. Under non voltage clamped conditions, H + gradient-dependent 22Na uptake into these vesicles was significantly inhibited by specific inhibitors of Na +-H + exchange (DMA, HMA and EIPA) as well as by inhibitor of epithelial Na + channels (phenamil). Under voltage clamped conditions, H + gradient-dependent 22Na uptake, however, was unaffected by phenamil (20 μM), but was almost completely inhibited by DMA, HMA and EIPA (20 μM each). The mechanism of amiloride inhibition of electroneutral Na +-H + exchange was noncompetitive with a K i for amiloride of 340 μM. Electroneutral 22Na uptake exhibited saturation kinetics with an apparent K m for Na + of 8.7 ± 1.7 mM and a V max of 2.02 ± 0.45 nmol/mg per 5 s. The Na +-H + exchange demonstrated cation specificity similar to the Na +-H + exchangers described in other epithelia. These studies demonstrate for the first time that Na + transport across the apical membranes of human distal colon involves both conductive Na + uptake and an electroneutral Na +-H + exchange process.