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

• Published in: American Journal of Physiology: Cell Physiology 2003-10, Vol.285 (4), p.C912-C921
• Main Authors: Ikuma, Mutsuhiro, Geibel, John, Binder, Henry J, Rajendran, Vazhaikkurichi M
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Animals; Chloride Channels - antagonists & inhibitors; Chloride-Bicarbonate Antiporters - metabolism; Chlorides - pharmacokinetics; Colon - metabolism; Fluoresceins; Hydrogen-Ion Concentration; Intracellular Membranes - metabolism; Male; Nitrobenzoates - pharmacology; Rats; Rats, Sprague-Dawley
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00396.2002

Departments of 1 Internal Medicine, and 2 Cellular and Molecular Physiology, and 3 Surgery, Yale University, New Haven, Connecticut 06520 Submitted 29 August 2002 ; accepted in final form 16 June 2003 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-HCO 3 exchange as the mechanism for 36 Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36 Cl 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-HCO 3 exchange. In the absence of voltage clamping, both outward OH gradient (pH out /pH in : 7.5/5.5) and outward HCO 3 gradient stimulated transient 36 Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36 Cl uptake, whereas HCO 3 gradient-driven 36 Cl uptake was only partially inhibited (38%). Both electroneutral HCO 3 and OH gradient-driven 36 Cl uptake were 1 ) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant ( K i ) of 26.9 and 30.6 µM, 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 12.6 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 HCO 3 -buffered and in the nominally HCO 3 -free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO 3 exchange is present in basolateral membranes and that, in the absence of HCO 3 , Cl-HCO 3 exchange can function as a Cl-OH exchange and regulate pH i across basolateral membranes of rat distal colon. crypt glands; superfusion; intracellular pH; membrane vesicles; 36 Cl uptake; Cl-anion exchange Address for reprint requests and other correspondence: V. M. Rajendran, Dept. of Internal Medicine, Yale Univ., 333 Cedar