title: Beta-adrenergic receptors couple to CFTR chloride channels of intercalated mitochondria-rich cells in the heterocellular toad skin epithelium.
abstract: In the heterocellular toad skin epithelium the beta-adrenergic receptor agonist isoproterenol activates cyclic AMP-dependent Cl(-) channels that are not located in the principal cells. With four experimental approaches, in the present study, we tested the hypothesis that the signalling pathway targets apical CFTR-chloride channels of mitochondria-rich cells. (i) Serosal application of isoproterenol (log(10)EC50=-7.1+/0.2; Hill coefficient=1.1+/0.2), as well as nor-adrenaline, activated an anion pathway with an apical selectivity sequence, G(Cl)>G(Br)> or =G(NO(3))>G(I), comparable to the published selectivity sequence of cloned human CFTR expressed in Xenopus oocytes. (ii) Known modulators of human CFTR, glibenclamide (200 micromol/l) and genistein (50 micromol/l), depressed and activated, respectively, the receptor-stimulated G(Cl). Genistein did not modify the anion selectivity. (iii) Transcellular voltage clamp studies of single isolated mitochondria-rich cells revealed functional beta-adrenergic receptors on the basolateral membrane. With approximately 60,000 mitochondria-rich cells per cm(2), the saturating activation of 11.9+/-1.6 ns/cell accounted for the measured isoproterenol-activated transepithelial conductance of 600-900 micros/cm(2). In forskolin-stimulated cells, glibenclamide (200 micromol/l) reversibly inhibited the transcellular conductance by 9.6+/1.6 ns/cell. (iv) A nucleotide sequence of one third of the Bufo bufo CFTR gene corresponding to the R-domain and part of the first nucleotide binding domain (NBD1) including its Walker motif was amplified from gallbladder epithelium. Somewhat smaller sequences of the BbCFTR were cloned from lung and isolated skin epithelium. The above new results taken together with our previously identified small-conductance CFTR-like Cl(-) channel in the apical membrane of isolated mitochondria-rich cells provide compelling evidence that the toad's CFTR gene codes for a functional Cl(-) channel in the apical plasma membrane of this minority cell type.
authors.affiliation: August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen, Denmark. ehlarsen@aki.ku.dk
authors.forename: Erik Hviid
authors.initials: EH
authors.lastname: Larsen
authors.forename: Jan
authors.initials: J
authors.lastname: Amstrup
authors.forename: Niels J
authors.initials: NJ
authors.lastname: Willumsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002023:Bufo bufo; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004594:Electrophysiology; D004847:Epithelial Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005712:Galvanic Skin Response; D019833:Genistein / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D011943:Receptors, Adrenergic, beta / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016415:Sequence Alignment; D012867:Skin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011943:Receptors, Adrenergic, beta; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019833:Genistein; D007545:Isoproterenol; D005905:Glyburide