Faculty Collaboration Database

Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells. Free Radic Biol Med 2003 Sep 15;35(6):662-75

Date

09/06/2003

Pubmed ID

12957658

DOI

10.1016/s0891-5849(03)00392-7

Scopus ID

2-s2.0-0042829388 (requires institutional sign-in at Scopus site)   11 Citations

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.

Author List

Thome U, Lazrak A, Chen L, Kirk MC, Thomas MJ, Forman HJ, Matalon S

Author

Michael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Chloride Channels
Chlorides
Electric Conductivity
Ion Transport
Male
Mass Spectrometry
Mice
Molecular Structure
Molsidomine
Patch-Clamp Techniques
Reactive Oxygen Species
Spectrometry, Mass, Electrospray Ionization
Trachea