2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

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.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chloride Channels; Chloride-Bicarbonate Antiporters; Chlorides; Colon; Fluoresceins; Hydrogen-Ion Concentration; Intracellular Membranes; Male; Nitrobenzoates; Rats; Rats, Sprague-Dawley

To examine the effect of hyperosmolality on Na(+)/H(+) exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH(i)) in a single MC from rat glomeruli. All the experiments were performed in CO(2)/HCO(-)(3)-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH(2)O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 microM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na(+). The hyperosmolality shifted the Na(+)-dependent acid extrusion rate vs. pH(i) by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular Cl(-) by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na(+)-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na(+)-dependent pH(i) recovery rate or Na(+)-dependent acid extrusion rate. The Cl(-)-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pH(i), 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl(-), and 4) the Cl(-)-dependent NHE activation under hyperosmotic conditions partly occurs via Cl(-) channel and microtubule-dependent processes.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Cell Size; Cells, Cultured; Chlorides; Colchicine; Fluoresceins; Glomerular Mesangium; Hydrogen-Ion Concentration; Hypertonic Solutions; Male; Nitrobenzoates; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchangers