Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for Cl-36 uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of Cl-36 uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pH(out)/pH(in): 7.5/5.5) and outward HCO3 gradient stimulated transient Cl-36 uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven Cl-36 uptake, whereas HCO3 gradient-driven Cl-36 uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven Cl-36 uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (K-i) of similar to26.9 and 30.6 muM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a K-m for Cl of similar to12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pH(i)) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein- acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pH(i) both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pH(i) across basolateral membranes of rat distal colon.