Mammalian Osmolytes and S-Nitrosoglutathione Promote ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein Maturation and Function

Abstract

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed ⌬F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of ⌬F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the ⌬F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that ⌬F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact ⌬F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted ⌬F508 CFTR maturation and function. S-Nitrosoglutathione-corrected ⌬F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting ⌬F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize ⌬F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why ⌬F508 homozygous patients do not present with kidney dysfunction.

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

“…In vitro strategies have included low temperature and chemical agents such as glycerol and 4-phenylbutyrate (22), as well as GSNO and other S-nitrosylating agents (18). Strikingly, previous studies (7,(14)(15)(16)(17)(18)(19) and the current work suggest that low micromolar GSNO concentrations increase maturation and cellsurface expression of ΔF508 CFTR. Here, we show that GSNO appears to act through a mechanism independent of-and additive with-low temperature to increase ΔF508 CFTR maturation and cell-surface expression.…”

“…(v) It can increase wild-type CFTR function (17). (vi) Last, it can increase ΔF508 expression and maturation (7,(14)(15)(16)(17)(18)(19), and the corrected protein is functional in monolayer and primary nasal cells (4, 7). Dose-response experiments in monolayer cell cultures suggest that concentrations of 5-10 μM may be optimal (15,16,18) (Fig.…”

“…S-nitrosoglutathione (GSNO) is an endogenous signaling molecule that relaxes airway smooth muscle (8)(9)(10), enhances ventilation-perfusion matching (9,10), increases ciliary beat frequency (11), has antimicrobial effects (12,13), and is one of a class of S-nitrosylating agents that increase expression, maturation, and function of wild-type (WT) and ΔF508 CFTR in primary nasal and monolayer cultures of epithelial cells (7,(14)(15)(16)(17)(18)(19). The GSNO effect on CFTR is partly transcriptional (16), but is primarily posttranscriptional (17,18).…”

Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses

“…For example, Welch and colleagues have suggested that stabilizing osmolytes, which they call 'chemical chaperones', might rescue misfolded proteins in human diseases (Welch and Brown, 1996). Recently, we found that addition of various mammalian osmolytes and TMAO can indeed restore function of one form of cystic fibrosis mutant protein ( Fig.·6; Howard et al, 2003). TMAO can also prevent misfolding of prion proteins .…”

Antioxidants in cystic fibrosis☆Conclusions from the CF Antioxidant Workshop, Bethesda, Maryland, November 11-12, 2003

“…Another potential benefit of augmenting intracellular GSH could be a reduction in Th2 response [67], resulting in a decrease in inflammation, as achieved in mice with P. aeruginosa lung infections [68,69]. Furthermore, increased GSH levels could augment levels of GSNO, a molecule that might improve maturation of CFTR and stimulate other chloride channels to increase chloride and GSH flux [70]. Unfortunately, there could also be dangers in altering the levels of such a pluripotent molecule.…”