title: Regulation of basolateral Cl(-) channels in airway epithelial cells: the role of nitric oxide.
abstract: The presence of basolateral Cl(-) channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl(-) conductance sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl(-) channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl(-) channels via a cGMP-dependent pathway. Anion replacement and (36)Cl(-) flux studies showed that NO affected both Cl(-) and HCO (3) (-) secretion. Two different types of Cl(-) channels are known to be present in the basolateral membrane of epithelial cells: Zn(2+)-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl(-) conductance in the presence of Zn(2+) ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl(-) conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl(-) channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.
authors.affiliation: Department of Physiology, University of Alberta, Edmonton AB, T6G 2H7, Canada.
authors.forename: Valentin
authors.initials: V
authors.lastname: Duta
authors.forename: Florentina
authors.initials: F
authors.lastname: Duta
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Puttagunta
authors.forename: A Dean
authors.initials: AD
authors.lastname: Befus
authors.forename: Marek
authors.initials: M
authors.lastname: Duszyk
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000578:Amidines / Q000494:pharmacology; D000838:Anions / Q000378:metabolism; D000873:Anthracenes / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D000075002:Bestrophins; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005136:Eye Proteins / Q000187:drug effects / Q000502:physiology*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D008168:Lung / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D000578:Amidines; D000838:Anions; D000873:Anthracenes; C113133:BEST1 protein, human; D001596:Benzylamines; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D020030:Nitric Oxide Donors; D010069:Oxadiazoles; D011810:Quinoxalines; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C010030:9-anthroic acid; D052247:Nitric Oxide Synthase Type II; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid