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

Abstract

Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Ci-/HCOi exchange and electrodiffusive HCOi transport in Necturus gallbladder epithelium, lntracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophyiline (3 x 10 -s M) or the adenylate cyclase activator forskolin (10 -5 M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H + and HCOL When the same experiment is performed after addition of amiloride or removal of Na + from the mucosal bathing medium, alkalinization is observed since H + transport is either inhibited or reversed, whereas HCOi secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular CIactivity (aCl 0 upon reducing mucosal solution [CI-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive CI-permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [CI-] and [HCOg] or lowering [C1-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by ~50%. Measurements of net CI-uptake upon increasing mucosal C1-from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external C1-was also inhibited significantly by theophylline. Reducing mucosal solution [HCO~] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCll. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (CI-/HCOg) exchange. Studies of the effects of rapid changes in mucosal [HCOg] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in Address reprint requests to Dr.

Effects of changes in mucosal solution Cl- or K+ concentration on cell water volume of Necturus gallbladder epithelium.

“…1 A and 2 A and summarized in Table I. In agreement with previous results, changes in cell membrane voltages and resistance ratio are small, and R t increased from 147 ---13 to 158 ± 13, P < 0.05 (Petersen and Reuss, 1983;Reuss, 1987). The concomitant changes in cell water volume and aCl( are also summarized in Table I Table I).…”

“…acid-fell from 15 -+ 1 to 7 + 1 mM with an initial rate of decline of 30.8 -+ 2.7 raM/rain. This rate is somewhat faster than previously reported, although the steady-state changes were similar (Reuss, 1987). Upon return to control Ringer's solution, both the cell water volume and the aCl~-returned to the values recorded before ion replacement.…”

“…However, if CI-enters the cells by anion exchange, then the fall in aCid-will be accompanied by an increase in aHCO~i and no change in cell volume. Reuss (1987) found that when mucosal [C1-] was reduced to 10 mM the calculated net efflux of C1-was equal to the calculated net influx of HCO~. If the osmotic and reflection coefficients for CI-and HCO~ are similar, the steady-state osmotic difference will be preserved and cell water volume will not change.…”

“…Patch-clamp studies have revealed the existence of a CI-channel in dissociated gallbladder cells stimulated by cAMP (Segal and Reuss, 1989). In addition, elevation of [cAMP] in Necturus gallbladder epithelium inhibits both apical membrane Na÷/H + and CI-/ HCO~ exchange by ~ 50% (Reuss and Petersen, 1985;Reuss, 1987).…”

“…The primary effects of elevation of cAMP in Necturus gallbladder epithelium include activation of an apical membrane CI-conductance and inhibition of both apical membrane Na+/H + and Cl-/HCO~ exchangers, with the net effect of reducing NaCI and fluid absorption (Petersen and Reuss, 1983;Reuss and Petersen, 1985;Reuss, 1987). Physiologically relevant hormonal regulation of ion transport by VIP and secretin via cAMP has been suggested for mammalian gallbladder (Wood and Svanvik, 1983;O'Grady et al, 1989).…”