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- conductanc e 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 biio nic potential measurements (SCN- > I- approximate to NO3- > Br- > Cl- much greater than ISE(-)) differed from the sequence derived from meas urements of apical membrane resistance (NO3- approximate to Br- approx imate to Cl- > SCN- > I- approximate to ISE(-)). Accordingly, the cond uctance was inhibited by SCN- and I- which, from the potential measure ments, appeared to be more permeable than Cl-. This finding agrees wit h observations of the cystic fibrosis transmembrane conductance regula tor (CFTR) Cl- channel reported recently. However, none of the commonl y used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-b enzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic aci d (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduce d this conductance in Necturus gallbladder. In contrast to the situati on in most other epithelia, elevation of intracellular Ca2+ concentrat ion ([Ca2+](i)) by ionomycin stimulated only K+ conductance and not th at 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 disr uption of the cytoskeleton by preincubation of the tissue with cytocha lasin D. The data indicate that Necturus gallbladder epithelium contai ns homologues of the CFTR Cl- channel which reside permanently in the apical cell membrane and which can be stimulated by a cAMP-dependent p hosphorylation process without involvement of cell Ca2+ or exocytosis.