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 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl 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 (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 microM, 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 Km for Cl of approximately 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) 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 pHi 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 pHi across basolateral membranes of rat distal colon.
Ikuma
Mutsuhiro
M
Department of Internal Medicine, Yale University, 333 Cedar St., New Haven, Connecticut 06520, USA.
Geibel
John
J
Binder
Henry J
HJ
Rajendran
Vazhaikkurichi M
VM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism*; D002712:Chlorides / Q000493:pharmacokinetics; D003106:Colon / Q000378:metabolism*; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D009579:Nitrobenzoates / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D018118:Chloride Channels; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D005452:Fluoresceins; D009579:Nitrobenzoates; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid