Regulation of membrane chloride currents in rat bile duct epithelial cells.
This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.
Fitz
J G
JG
Duke University and Veterans Administration Medical Center, Durham, NC 27710.
Basavappa
S
S
McGill
J
J
Melhus
O
O
Cohn
J A
JA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D003550:Cystic Fibrosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D007454:Iodides / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis* / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013873:Thionucleotides / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D007454:Iodides; D008565:Membrane Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D015759:Ionomycin; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium