Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1 (original) (raw)
Summary
Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2_H_-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagent_para_-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.
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Abbreviations
WST-1:
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2_H_-tetrazolium, monosodium salt
MTT:
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
XTT:
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2_H_-tetrazolium, monosodium salt
MTS:
3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymemoxyphenyl)-2-(4-sulfophenyl)-2_H_-tetrazolium, inner salt
TTFA:
thenoyltrifluoroacetone
pCMBS:
_p_-chloromercuriphenylsul-fonic acid
SOD:
Superoxide dismutase
PMOR:
plasma membrane
NADH:
oxidoreductase
PMS:
phenazine methosulfate
PMA:
phorbol myristate acetate
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- Malaghan Institute of Medical Research, Wellington School of Medicine, P.O. Box 7060, Wellington South, New Zealand
M. V. Berridge & A. S. Tan
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- M. V. Berridge
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Berridge, M.V., Tan, A.S. Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1.Protoplasma 205, 74–82 (1998). https://doi.org/10.1007/BF01279296
- Received: 07 May 1998
- Accepted: 27 August 1998
- Issue Date: March 1998
- DOI: https://doi.org/10.1007/BF01279296