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 IC₅₀ of 3.2 ± 1 μm (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 μm) 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 μm), 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 μm). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.