PURPOSE: The endogenous bronchodilator, S-nitrosoglutathione (GSNO) has been proposed as a possible pharmacological remedy that reverses the delF508-CFTR maturation defect and increases CFTR-mediated chloride efflux in CF airway epithelial cells (AEC). Previously, we have confirmed the activation of chloride efflux by GSNO in primary nasal epithelial cells from CF patients homozygous for the delF508 CFTR mutation. The effect of GSNO is dependent on intracellular SNO trafficking. Zhang and Hong (2004) reported that presence of L-cystine enhanced GSNO-dependent S-nitrosothiol uptake and increased the intracellular S-nitrosothiol level. The present study sought to determine whether L-cystine would augment the effect of GSNO on chloride efflux from CF AEC.
METHODS: The effects of 10 μM GSNO at various incubation times in a combination with L-cystine or alone were investigated on CF AEC cell line: bronchial surface epithelium (CFBE41o-). Intracellular chloride concentrations were measured using fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE).
RESULTS: The treatment with 10 μM GSNO combined with 20 μM L-cystine (Lcys) resulted in significantly increased chloride efflux (1.25 mM/s) from CFBE cells after 5 minutes exposure comparing to control efflux rate (0.68 mM/s). The increased efflux was sensitive to DIDS (a non-specific blocker of chloride channels other than CFTR). Chloride efflux rates in these cells after 1h and 4h exposure of GSNO+Lcys were not different from the control (0.96 and 0.75 mM/s respectively). L-cystine alone had no effect on chloride transport in CF cells (Fig.1). The treatment with 10 μM GSNO increased chloride transport in CFBE cells after 4h incubation (Fig.2) but not with shorter incubation (5 min, 30 min and 1 h). However, the effect was present for longer time if incubation continued (4 - 6h tested).
CONCLUSION: L-cystine influences GSNO effect on chloride transport in AEC: a combination of GSNO with L-cystine increases chloride transport in CFBE cells after 30 min incubation but effect is rather short-lived.
CLINICAL IMPLICATIONS: GSNO has been proposed as a potential therapy for CF.
DISCLOSURE: Zhanna Servetnyk, No Financial Disclosure Information; No Product/Research Disclosure Information