Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder.

Abstract

AB S T R A C T The hypothesis of Cl -/HCO s exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pH.), intracellular pH (pHi), and Cl -activity (aCli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na'/H' exchange inhibitor amiloride (10-3 M) to the superfusate, alkalinization was observed . During prolonged (10 min) exposure to amiloride, no significant pH . change occurred . Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na' in the superfusate (TMA' substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of CI -(cyclamate or sulfate substitution) caused a shortlived increase in the rate of luminal acidification, followed by a return to control values (10-30 min) . Upon re-exposure to Cl -, there was a transient reduction of luminal acidification . The initial increase in acidification produced by CIremoval was partially inhibited by SITS (0 .5 mM). The pH i increased rapidly and reversibly when the Cl-concentration o£ the mucosal bathing solution was reduced to nominally 0 mM . The pH; changes were larger in 10 mM HC03-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO-3 is transported in exchange for CI -. In both HEPES-and HC03-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of aCli. These results support the hypothesis of apical membrane CI -/HC03 exchange, which can be dissociated from Na''/H+ exchange and operates under control conditions . The coexistence at the apical membrane of Na'/H' and CI -/HCO3 antiports suggests that NaCl entry can occur through these transporters .

Cyclic AMP inhibits Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder epithelium.

“…From the initial rate of change of the extracellular pH and the buffering power ofthe solution, an apparent H+ flux (OH) was computed and normalized to that determined under control conditions (NaCl HEPES-Ringer's, before theophylline) . In the absence of theophylline, both amiloride and Na'-free medium reversed the luminal acidification to luminal alkalinization, as previously reported (Reuss and Costantin, 1984). The larger alkalinization observed in Na*-free medium compared with amiloride is due to reversal of the Na''/H+ exchange in the former condition .…”

“…In parallel with these studies, we demonstrated the existence of an Na'/H' exchanger at the apical membrane of Necturus gallbladder epithelium, whose rate under control conditions accounts for at least 75% of Na' entry Reuss, 1982, 1984;Reuss, 1984). This cation exchanger operates in parallel with a Cl-/ HC03 exchanger (Reuss and Costantin, 1984), which in turn accounts for the rate of transepithelial Cl-transport (Reuss, 1984).…”

“…(b) It cannot be ascribed to an H+ influx (lumen to cell), because in tissues exposed to 10' M amiloride in the presence of 100 mM Na', the net H' flux, although reduced, is from cell to lumen (Weinman and Reuss, 1984;Reuss, 1984). (c) Under conditions comparable to those in the pHo experiments, the changes in pHi and aCli are consistent with electroneutral anion exchange (Reuss and Costantin, 1984;this article) .…”

“…The pH of the mucosal bathing solution (volume, was measured with a small glass electrode upon stopping superfusion . To estimate HCO; secretion across the apical membrane, the control solution was rapidly replaced with Na'`-free medium (TMA+ substitution) or with HEPES-Ringer's plus 10' M amiloride, and superfusion was stopped (see Reuss and Costantin, 1984) . The buffering powers of the solutions were determined by titration .…”

“…At an external [HC03] of 25 mM, the intracellular HCO3 concentration would be 51 .8 mM, and pH; (assuming equal values of Pco= in extra-and intracellular phases) would be, at most, 6.26. This possibility is unlikely on the basis of pHi measurements in Necturus gallbladder under a variety of experimental conditions (Weinman and Reuss, 1982;Reuss and Costantin, 1984;Reuss and Petersen, 1985). Only during acid-loading experiments by the ammonium chloride prepulse technique (see Boron and De Weer, 1976) did the pH; reach values as low as 6.5 (Reuss and Petersen, 1985).…”