Potentiometric measurement of intracellular redox activity

Living cells typically contain numerous redox couples such as NAD(+)/NADH, NADP(+)/NADPH, cystine/cysteine, and the oxidized and reduced forms of glutathione and metalloenzymes. It is expected that the intracellular concentrations of these couples may change selectively with changes in the cellular environment, such as those due to hormones, growth factors, and nutrients. The present work employs an electrochemical potentiometric means of probing intracellular redox activity in live cells. The couple menadione/menadiol is used as a carrier mediator, carrying electrons across the cell membrane, and sensing intracellular redox enzyme activity. The extracellular menadiol redox activity is monitored by means of an extracellular solution containing the ferricyanide/ferrocyanide couple and a gold electrode. Evidence is presented that the extracellular rate of reduction of ferricyanide is limited by the intracellular rate of reduction of menadione. A substantial fraction (30-80% depending on cell type) of ferricyanide reduction is mediated by DT-diaphorase-catalyzed transfer of electrons from NADPH to menadione. Reduction of menadione and oxidation of menadiol by enzymes of the mitochondrial electron transport chain also contribute to the observed ferricyanide reduction rate. Some membrane receptor agonists increase the ferricyanide reduction rate, whereas others decrease this rate, demonstrating specific hormonal regulation of cellular redox enzyme activity.