title: Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells.
abstract: Amiloride-sensitive epithelial Na(+) channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability (Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative (n = 21; P < 0.05). Treatment of 400 μM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 μM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo.
authors.affiliation: Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, Georgia;
authors.forename: Charles A
authors.initials: CA
authors.lastname: Downs
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia;
authors.forename: Lisa
authors.initials: L
authors.lastname: Kreiner
authors.affiliation: Department of Computer Science, School of Electronics and Information Engineering, Tongji University, Shanghai, China.
authors.forename: Xing-Ming
authors.initials: XM
authors.lastname: Zhao
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia;
authors.forename: Phi
authors.initials: P
authors.lastname: Trac
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia;
authors.forename: Nicholle M
authors.initials: NM
authors.lastname: Johnson
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia; Center for Cystic Fibrosis and Airways Disease Research at Children's Healthcare of Atlanta Hospital, Atlanta, Georgia; and.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Hansen
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia; Center for Cystic Fibrosis and Airways Disease Research at Children's Healthcare of Atlanta Hospital, Atlanta, Georgia; and.
authors.forename: Lou Ann
authors.initials: LA
authors.lastname: Brown
authors.affiliation: Department Pediatrics, School of Medicine, Emory University, Atlanta, Georgia; Center for Cystic Fibrosis and Airways Disease Research at Children's Healthcare of Atlanta Hospital, Atlanta, Georgia; and mhelms@emory.edu.
authors.forename: My N
authors.initials: MN
authors.lastname: Helms
mesh_terms: D062565:Acid Sensing Ion Channels / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005260:Female; D019803:Glutathione Disulfide / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D018408:Patch-Clamp Techniques; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000378:metabolism*
chemical_list: D062565:Acid Sensing Ion Channels; D000975:Antioxidants; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D006861:Hydrogen Peroxide; D019803:Glutathione Disulfide
keywords: Cys thiol; alveolar flooding; oxidative stress; redox potential