Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells

Academic Article

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 Ω/cm2 and then were mounted in Ussing chambers. Baseline short-circuit current (ISC) values, prior to and following the addition of 10 μM amiloride (an inhibitor of sodium-transport pathways) into the apical side, were 65 ± 1.9 μA/cm2 and 7.6 ± 0.51 μA/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 μA/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 μM), an inhibitor of cystic fibrosis transmembrane regulator, or reduced by glutathione (GSH, 5 mM), a scavenger of peroxynitrite. Forskolin (10 μM) 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 μM. 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. © 2003 Elsevier.
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    Digital Object Identifier (doi)

    Author List

  • Thome U; Lazrak A; Chen L; Kirk MC; Thomas MJ; Forman HJ; Matalon S
  • Start Page

  • 662
  • End Page

  • 675
  • Volume

  • 35
  • Issue

  • 6