CALCIUM IS NOT INVOLVED IN THE CAMP-MEDIATED STIMULATION OF CL- CONDUCTANCE IN THE EPICAL MEMBRANE OF NECTURUS GALLBLADDER EPITHELIUM

The permeability properties of the forskolin-stimulated Cl- conductance in the apical membrane of Necturus us gallbladder epithelium and the possible participation of intracellular Ca2+ in its stimulation have been investigated. The anion selectivity sequence as derived from biionic potential measurements (SCN- > I- approximate to NO3- > Br- > Cl- much greater than ISE(-)) differed from the sequence derived from measurements of apical membrane resistance (NO3- approximate to Br- approximate to Cl- > SCN- > I- approximate to ISE(-)). Accordingly, the conductance was inhibited by SCN- and I- which, from the potential measurements, appeared to be more permeable than Cl-. This finding agrees with observations of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel reported recently. However, none of the commonly used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduced this conductance in Necturus gallbladder. In contrast to the situation in most other epithelia, elevation of intracellular Ca2+ concentration ([Ca2+](i)) by ionomycin stimulated only K+ conductance and not that of Cl- in the apical cell membrane. Chelation of intracellular Ca2+ did not prevent the stimulation of Cl- conductance by forskolin. This indicates that [Ca2+](i) does not have even a permissive role in the cyclic adenosine monophosphate-(cAMP)-mediated stimulation process, as would have been expected if exocytosis was involved. Further evidence against the involvement of exocytosis in the stimulation process came from the observation that the stimulation was not associated with an increase in apical membrane capacitance and was not suppressed by disruption of the cytoskeleton by preincubation of the tissue with cytochalasin D. The data indicate that Necturus gallbladder epithelium contains homologues of the CFTR Cl- channel which reside permanently in the apical cell membrane and which can be stimulated by a cAMP-dependent phosphorylation process without involvement of cell Ca2+ or exocytosis.