MECHANISMS OF NA+ TRANSPORT IN HUMAN DISTAL COLONIC APICAL MEMBRANE-VESICLES

Apical membrane vesicles purified from mucosal scrapings obtained from distal segments of organ donor colons and a Na-22-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 Na-22 into these vesicles. Osmolarity studies demonstrated that Na-22 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 Na-22 uptake into these vesicles by similar to 45% and generation of an inside negative membrane potential significantly increased Na-22 uptake. Under non voltage clamped conditions, H+ gradient-dependent Na-22 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 Na-22 uptake, however, was unaffected by phenamil (20 mu M), but was almost completely inhibited by DMA, HMA and EIPA (20 mu M each). The mechanism of amiloride inhibition of electroneutral Na+-H+ exchange was noncompetitive with a K-i for amiloride of 340 mu M. Electroneutral Na-22 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.