The effects of tert-butyl hydroperoxide on human erythrocyte membrane ion transport and the protective actions of antioxidants.
The oxidising actions of tert-butyl hydroperoxide (tBH) (0-3 mmol/l) on human erythrocyte membrane ion transport have been studied using measurements of 86Rb+ influx. Ouabain and bumetanide were used to distinguish Rb+ flux via the sodium pump (Na,K-ATPase), Na,K,2Cl cotransporter and through residual membrane permeability. The protective actions of antioxidants and related molecules (vitamin E, vitamin E acetate, Trolox, butylated hydroxytoluene (BHT) and dithioerythritol (DTE) were studied. The effects of tBH were concentration dependent and the mean residual (ouabain and bumetanide insensitive) Rb+ permeability was increased by a factor of 8.5 (S.E.M. 2.2, n = 15) by a 5-min exposure to 2 mmol/l tBH. This action was almost completely prevented by co-incubation with Trolox or BHT, and partially prevented by the presence of vitamin E or DTE. Incubation with 2 mmol/l tBH for 5 min increased intracellular Na+ by a factor of 1.8 (S.E.M. 0.1, n = 8) and reduced intracellular K+ by a factor of 0.93 (S.E.M. 0.03, n = 8). These effects were prevented by Trolox and partially prevented by vitamin E, whereas DTE and vitamin E acetate were ineffective. Incubation with 2 mmol/l tBH for 5 min reduced the mean apparent sodium pump Vmax by 43% (S.E.M. 4, n = 8). This effect was completely prevented by Trolox and partially prevented by vitamin E. Vitamin E acetate had no effect. The mean bumetanide-sensitive Rb+ influx via the Na,K,2Cl cotransporter was reduced by 30% (S.E.M. 8.7, n = 25) by a 5-min exposure to 2 mmol/l tBH. This action was variable and no significant actions of the antioxidants studied could be demonstrated. This study suggests that tBH-mediated oxidative damage occurs from a hydrophilic site and involves increased non-selective membrane cation permeability and inhibition of specific transport systems.
Dwight
J F
JF
Department of Cardiology, Southmead Hospital, Bristol, UK.
Hendry
B M
BM
D000975:Antioxidants / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood; D002839:Chromans / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000097:blood / Q000187:drug effects*; D010042:Ouabain / Q000494:pharmacology; D010545:Peroxides / Q000494:pharmacology*; D012413:Rubidium / Q000097:blood; D015222:Sodium Channels / Q000097:blood / Q000187:drug effects; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood; D024505:Tocopherols; D014810:Vitamin E / Q000031:analogs & derivatives / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000031:analogs & derivatives*; D020122:tert-Butylhydroperoxide
D000975:Antioxidants; D002352:Carrier Proteins; D002839:Chromans; D007473:Ion Channels; D010545:Peroxides; D015222:Sodium Channels; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D014810:Vitamin E; D002084:Butylated Hydroxytoluene; D010042:Ouabain; D004226:Dithioerythritol; D020122:tert-Butylhydroperoxide; D000254:Sodium-Potassium-Exchanging ATPase; D024502:alpha-Tocopherol; D012413:Rubidium; D024505:Tocopherols; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid