Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells. Free Radic Biol Med 2003 Sep 15;35(6):662-75
Date
09/06/2003Pubmed ID
12957658DOI
10.1016/s0891-5849(03)00392-7Scopus ID
2-s2.0-0042829388 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
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.
Author List
Thome U, Lazrak A, Chen L, Kirk MC, Thomas MJ, Forman HJ, Matalon SAuthor
Michael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsChloride Channels
Chlorides
Electric Conductivity
Ion Transport
Male
Mass Spectrometry
Mice
Molecular Structure
Molsidomine
Patch-Clamp Techniques
Reactive Oxygen Species
Spectrometry, Mass, Electrospray Ionization
Trachea