Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione.
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.
Chen
Lan
L
Department of Anesthesiology, University of Alabama, Birmingham, Alabama 35233, USA.
Patel
Rakesh P
RP
Teng
Xinjun
X
Bosworth
Charles A
CA
Lancaster
Jack R
JR
Matalon
Sadis
S
D001993:Bronchodilator Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000493:pharmacokinetics*; D006152:Cyclic GMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D004594:Electrophysiology; D006801:Humans; D018832:Molecular Chaperones; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D026361:Reactive Nitrogen Species; D020545:Respiratory Mucosa / Q000166:cytology; D026422:S-Nitrosoglutathione / Q000494:pharmacology*
D001993:Bronchodilator Agents; D018118:Chloride Channels; D002712:Chlorides; D018832:Molecular Chaperones; D026361:Reactive Nitrogen Species; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D006152:Cyclic GMP