title: Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells.
abstract: We examined the effects of reactive oxygen-nitrogen intermediates on chloride (Cl-) currents across murine tracheal epithelial (MTE) cells isolated from CD-1 mice. MTE cells were cultured on permeable supports until they formed water-tight monolayers with transepithelial resistances (Rt)>500 Omega/cm2 and then were mounted in Ussing chambers. Baseline short-circuit current (ISC) values, prior to and following the addition of 10 microM amiloride (an inhibitor of sodium-transport pathways) into the apical side, were 65 +/- 1.9 microA/cm2 and 7.6 +/- 0.51 microA/cm2, respectively (X +/- 1 SE, n=32). The addition of 3-morpholinosydnominine (SIN-1, 1 mM), which generates both superoxide and nitric oxide anions, to amiloride-treated monolayers resulted in a transient increase of ISC to a peak value of 35 +/- 1.3 microA/cm2 (X +/- SE, n=14) within the next 30-60 min. After this, the ISC decreased gradually and returned to its pre-SIN-1 value. These changes were blocked by glibenclamide (200 microM), an inhibitor of cystic fibrosis transmembrane regulator, or reduced by glutathione (GSH, 5 mM), a scavenger of peroxynitrite. Forskolin (10 microM) augmented the SIN-1 effect when added at the peak of the SIN-1 response but not when ISC had returned to its baseline value. Perfusion of MTE cells with SIN-1 also increased whole cell Cl- currents 4-fold and the open probability of CFTR-type single-channel currents from 0.041 to 0.92 in a transient fashion. Decomposed SIN-1, but not pure SIN-1c (the stable decomposition product of SIN-1), also increased ISC with an EC50 of 5 microM. Electrospray mass spectroscopy revealed several unique and uncharacterized compounds formed during the decomposition of SIN-1 as well as the reaction of SIN-1c with peroxynitrite. Formation of these compounds was inhibited by GSH. We conclude that compounds formed by the reaction of peroxynitrite with by-products of SIN-1, rather than reactive oxygen-nitrogen species per se, were responsible for the modulation of Cl- secretion across primary cultures of MTE cells.
authors.affiliation: Division of Neonatology, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294-2172, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Thome
authors.forename: Ahmed
authors.initials: A
authors.lastname: Lazrak
authors.forename: Lan
authors.initials: L
authors.lastname: Chen
authors.forename: Marion C
authors.initials: MC
authors.lastname: Kirk
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Thomas
authors.forename: Henry Jay
authors.initials: HJ
authors.lastname: Forman
authors.forename: Sadis
authors.initials: S
authors.lastname: Matalon
mesh_terms: D000818:Animals; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D004553:Electric Conductivity; D017136:Ion Transport; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017382:Reactive Oxygen Species / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014132:Trachea / Q000166:cytology* / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D017382:Reactive Oxygen Species; C002385:linsidomine; D008981:Molsidomine