Electron Current and Proton Current in Activated Human Monocytes - Strong Glucose Dependence of the Electron Current

Abstract

Monocytes play multiple roles in the immune system, among other things, linking innate to adaptive immunity. Despite their biological importance, monocytes alone among all other phagocytes have not been investigated during the “respiratory burst” - the coordinated activation of NADPH oxidase and proton channels. Voltage gated proton current and NADPH oxidase generated electron current were measured simultaneously in human monocytes in perforated patch configuration after one to three days in culture. Upon stimulation by 60 nM phorbol myristate acetate (PMA), electron current (reflecting NADPH oxidase activity) appeared and the proton current amplitude increased. PMA slowed the kinetics of tail currents, sped the activation of outward proton current, and shifted the gH-V relationship negatively. The NADPH oxidase inhibitor diphenylene iodonium (DPI) inhibited the electron current but affected solely the tail current kinetics of the proton current. Thus, although monocytes differentiate from a different lineage than granulocytes, their responses to PMA resemble those of other phagocytes. Hydrogen peroxide (H2O2) production was recorded fluorometrically. Zinc, at concentrations that inhibit proton current, reduced H2O2 production in monocytes. This Zn2+ sensitivity resembles that seen in neutrophils and eosinophils. The dogma that extracellular glucose is necessary to support the oxidative burst of human monocytes was tested in electrophysiological and fluorescence measurements. Electron current measured in patch clamp experiments was increased 2.5 fold by adding glucose to the bath solution; proton current was unaffected. Correspondingly, H2O2 production was strongly increased and more sustained in the presence of glucose. In summary, the electrophysiological events during activation of monocytes resemble those in other phagocytes, but NADPH oxidase is more acutely dependent on the presence of glucose.