title: GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells.
abstract: Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO(2))-mediated oxidation, diazene dicarboxylic acid bis-N,N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO(2) > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO(2) and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.
authors.affiliation: Laboratory of Pathobiochemistry, School of Veterinary Medicine, Azabu University, Fuchinobe, Sagamihara, Kanagawa 229, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Fujise
authors.forename: K
authors.initials: K
authors.lastname: Higa
authors.forename: T
authors.initials: T
authors.lastname: Kanemaru
authors.forename: M
authors.initials: M
authors.lastname: Fukuda
authors.forename: N C
authors.initials: NC
authors.lastname: Adragna
authors.forename: P K
authors.initials: PK
authors.lastname: Lauf
mesh_terms: D000818:Animals; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004285:Dogs; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology; D010084:Oxidation-Reduction; D011188:Potassium / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D002712:Chlorides; D004137:Dinitrochlorobenzene; D007202:Indicators and Reagents; D009566:Nitrates; D013439:Sulfhydryl Reagents; D003958:Diamide; C031618:sodium nitrate; D005978:Glutathione; D011188:Potassium