Chemical and pathological oxidative influences on band 3 protein anion-exchanger.
OBJECTIVE
The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells.


METHODS
To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration.


RESULTS
In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH.


CONCLUSIONS
The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation.
Teti
Diana
D
Department of Experimental Pathology and Microbiology, Section of Experimental Pathology, Faculty of Medicine and Surgery, Messina, Italy.
Crupi
Maria
M
Busá
Maria
M
Valenti
Andrea
A
Loddo
Saverio
S
Mondello
Maurizio
M
Romano
Leonardo
L
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D048429:Cell Size; D002712:Chlorides / Q000097:blood; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000097:blood; D005978:Glutathione / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010766:Phosphorylation; D011188:Potassium / Q000097:blood; D013431:Sulfates / Q000097:blood; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters / Q000097:blood / Q000737:chemistry; D000096922:K Cl- Cotransporters
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D005978:Glutathione; D008274:Magnesium; D016877:Oxidants; D011188:Potassium; D013431:Sulfates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D027981:Symporters; D000096922:K Cl- Cotransporters