Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Abstract

Enhanced cellular cAMP levels have been shown to increase apical membrane Cl- and HCO3- conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels in Necturus gallbladder. We used conventional open-tip and double-barreled Cl- -selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl- activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23 degrees C and pH 7.4. In HCO3- -free media, 0.1 mM IBMX added to the mucosal medium depolarized the apical membrane potential Va, decreased the fractional resistance FR, and significantly reduced intracellular Cl- activity (aCli). Under control conditions, aCli was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25 mM HCO3-, IBMX caused a small transient hyperpolarization of Va followed by a depolarization not significantly different from that observed in HCO3- -free Ringer's. Removal of mucosal Cl-, Na+ or Ca2+ did not affect the IBMX-induced depolarization in Va. The basolateral membrane of Necturus gallbladder is highly K+ permeable. Increasing serosal K+ from 2.5 to 80 mM, depolarized Va. Mucosal IBMX significantly reduced this depolarization. Addition of 10 mM Ba2+, a K+ channel blocker, to the serosal medium depolarized Va and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO3- conductance and decreases basolateral K+ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization of Va.

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

“…The results presented above indicate that, in Necturus gallbladder epithelium, elevation of intracellular cAMP inhibits apical membrane Cl-/HC03 exchange, but does not induce a sizable apical membrane electrodiffusive HCO3 permeability, a possibility previously suggested by others (Zeldin et al, 1985). Additional evidence for stimulation of an apical membrane Cl--conductive pathway by cAMP has been presented.…”

“…Second, the change in apical membrane voltage in response to luminal ionic substitutions becomes closer to the change in zero-current voltage when the apical membrane conductance is elevated; i.e., an indirect effect of [HC03] could cause a larger change in V., simply because of the lower value of Ra/Rb in theophylline . If the primary effect of cAMP were to reduce basolateral PK and hence to increase Rb (Zeldin et al, 1985), the same argument could be made.…”

“…Second, the elevation of aCli could be underestimated by conductive efflux of the Cl-that enters via anion exchange . Because of the recent conclusions of Zeldin et al (1985), the possibility of a cAMP-dependent apical membrane electrodiffusive HCO3 permeability was further examined . If cAMP induces or enhances an apical membrane HC0j conductance, then reducing luminal [HC03] should cause a greater cell membrane depolarization than under control conditions .…”

“…Arguments against Induction ofan HCO3-conductive Pathway Zeldin et al (1985) argued that elevation of intracellular cAMP, induced in Necturus gallbladder epithelium by exposure to isobutylmethylxanthine (IBMX), produced an increase in apical membrane HC03 conductance. They showed that in a Cl--free, HC03-free medium, IBMX produced monotonic cell membrane depolarization ; however, in the presence of 25 mM HCO3 in the bathing solutions (at constant pH), IBMX caused a transient hyperpolarization, followed by depolarization .…”

“…This suggestion was based on the observation of a reduction, in theophylline-treated preparations, of the secretion of base equivalents into the mucosal solution observed shortly after exposure to 1 mM amiloride. Zeldin et al (1985) recently suggested that CAMP might also elicit an HCOs -conductive pathway at the apical membrane of Necturus gallbladder. We have argued that these conclusions are incorrect (Reuss and Stoddard, 1987 ;Reuss, 1987).…”