Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells.
Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40-60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.
Tan
An S
AS
Malaghan Institute of Medical Research, Wellington, New Zealand.
Berridge
Michael V
MV
D020003:1-Octanol / Q000494:pharmacology; D015086:2,6-Dichloroindophenol / Q000378:metabolism*; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004396:Coloring Agents / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology*; D005292:Ferricyanides / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008773:Methylphenazonium Methosulfate / Q000031:analogs & derivatives* / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D004396:Coloring Agents; D005292:Ferricyanides; D013778:Tetrazolium Salts; D014451:Ubiquinone; D000625:Aminooxyacetic Acid; D008773:Methylphenazonium Methosulfate; C023530:1-methoxy-5-methylphenazinium methyl sulfate; D015086:2,6-Dichloroindophenol; D013482:Superoxide Dismutase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D020003:1-Octanol; C028033:potassium ferricyanide