Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium.
Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl 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 GaCl or interfered with activation of GaCl by forskolin. N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl 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 the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl 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 GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.
Heming
T A
TA
Department of Physiology, University of Texas Medical Branch, Galveston 77555.
Copello
J
J
Reuss
L
L
D000818:Animals; D002118:Calcium / Q000502:physiology; D018118:Chloride Channels / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology; D004789:Enzyme Activation / Q000502:physiology; D004848:Epithelium / Q000378:metabolism; D004988:Ethers, Cyclic / Q000494:pharmacology; D005704:Gallbladder / Q000378:metabolism*; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009337:Necturus; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013806:Theophylline / Q000494:pharmacology
D018118:Chloride Channels; D004988:Ethers, Cyclic; D005576:Colforsin; D019319:Okadaic Acid; C016276:8-bromocyclic GMP; D015759:Ionomycin; D013806:Theophylline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D006152:Cyclic GMP; D002118:Calcium