REGULATION OF CAMP-ACTIVATED APICAL MEMBRANE CHLORIDE CONDUCTANCE IN GALLBLADDER EPITHELIUM

Regulation of the cAMP-activated apical membrane Cl- conductance (G(Cl)(a)) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. G(Cl)(a) was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on G(Cl)(a) or interfered with activation of G(Cl)(a) by forskolin. N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the G(Cl)(a) response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated G(Cl)(a) but had no effects on intracellular [cAMP]. G(Cl)(a) was unaffected by 4 alpha-phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed ed the activation of G(Cl)(a) by 8-Br-cAMP, hastened the return of G(Cl)(a) to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the G(Cl)(a) changes elicited by theophylline. We conclude that: (a) NGB G(Cl)(a) can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) G(Cl)(a) can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of G(Cl)(a); OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects G(Cl)(a) activation, and (d) the apical membrane of NGB epithelium lacks a Ca2+-activated Cl- conductance.