Chloride transport in human proximal colonic apical membrane vesicles

The mechanism(s) of Cl- transport across the human colonic apical membranes are not well understood. Apical membrane vesicles (AMV) purified from organ donor proximal colonic mucosa and a rapid millipore filtration technique were utilized to study Cl-36(-) uptake into these vesicles. Outwardly directed OH- and HCO3- gradient stimulated Cl- uptake into these vesicles demonstrating a transient accumulation over equilibrium uptake. Voltage clamping the membrane potential of the vesicles or making them inside positive with K+/valinomycin failed to influence chloride uptake, indicating that the conductive Cl- uptake pathway is minimal in proximal colonic AMV. Anion exchange inhibitors, DIDS and SITS (1 mM) inhibited OH- and HCO3- stimulated Cl-36(-) uptake by approximate to 60%. Furosemide also demonstrated a small but significant inhibition of chloride uptake. Amiloride, bumetanide and acetazolamide (1 mM) failed to inhibit Cl-36 uptake. HCO3- and pH gradient stimulated 36 Cl- uptake exhibited saturation kinetics with an apparent K-m for chloride of 4.0 +/- 0.7 mM and a V-max of 17.8 +/- 3.9 nmol/mg per min. Bromide, chloride, nitrate and acetate (50 mM each) inhibited 5 mM Cl-36 uptake. Inwardly directed gradients of Na+, K+, or Na+ and K+ did not stimulate Cl-36(-) uptake into these vesicles, indicating that uptake of Na+ and Cl- in human proximal colonic AMV does not involve Na-Cl or Na-K-2Cl cotransport. The above findings indicate that chloride transport in human proximal colonic AMV involves an electroneutral Cl--HCO3- (OH-) exchange process, In view of the previous demonstration of a Na+-H+ antiporter in these vesicles, dual ion exchange mechanism of Na+-H+ and Cl--HCO3- in apical membrane domain of human colonocytes is postulated to be the primary mechanism for NaCl absorption in the human proximal colon.