Abstract
We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl(-)) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I(sc)) and trans-epithelial resistance (R(t)). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I(sc) with an IC(50) of 3.2 +/- 1 mum (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I(sc). Conversely, BAY 41-2272, a sGC stimulator, increased I(sc) in a dose-response fashion. The GSNO increase of I(sc) was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mum) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I(sc). However, smaller concentrations of oxy-Mb (1-10 mum), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I(sc) partially. Oxy-Mb did not reverse the increase of I(sc) following addition of GSNO and cysteine (50 mum). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.
Highlights
There is considerable evidence that reactive oxygen nitrogen species, generated by environmental pollutants and activated inflammatory or airway cells, may alter the properties of a number of ion channels either by direct modification or via signal transduction pathways [7]
Generation of NO—Addition of 10 M GSNO into the apical compartment of an Ussing chamber containing empty filters resulted in the immediate formation of NO up to ϳ1 M, which declined to a steady-state value of ϳ0.3 M, lasting over 45 min
The time course of Isc following apical addition of GSNO paralleled the NO formation profiles in the Ussing chamber as measured by an NO electrode
Summary
There is considerable evidence that reactive oxygen nitrogen species, generated by environmental pollutants and activated inflammatory or airway cells, may alter the properties of a number of ion channels either by direct modification (such as nitrosation, nitration, and oxidation) or via signal transduction pathways [7]. Our previous studies indicate that prolonged exposure of confluent monolayers of Calu-3, 16 human bronchial epithelial, or mouse tracheal epithelial cells to physiological levels of NO, generated by the chemical donor DETA NONOate, decrease levels of CFTR in their apical membranes and impaired chloride (ClϪ) secretion in response to cAMP, a well established regulator of CFTR [8, 9] These changes were found to be due to post-translational modification of CFTR by reactive oxygen nitrogen intermediates. Wang et al [16] reported that oxidized forms of glutathione (including GSNO) decreased CFTR function by oxidative modifications (glutathionylation) None of these studies utilized confluent monolayers of airway cells and were unable to ascertain the effects of GSNO on vectorial ClϪ transport. Activation of CFTR by GSNO increases were partly due to (i) stimulation of soluble guanylyl cyclase and possible phosphorylation of CFTR via cGMP-dependent mechanisms and (ii) nitrosation of CFTR or other chaperon proteins by reactive intermediates of GSNO and cysteine (S-nitrocysteine), entering the cytoplasm via amino acid transport system L, where they are capable of nitrosating proteins without the intermediate formation of NO [20]
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