Measurement of superoxide release from single pulmonary alveolar macrophages

An electroptical method was developed to quantify superoxide (O₂^-) release from single rat pulmonary alveolar macrophages (PAM) during adherence to the bottom of a culture dish. This was done by measuring the reduction of nitro blue tetrazolium (NBT) to a diformazan precipitate at 550 nm from videorecorded images of individual cells. Temporal changes in cell optical density, which are proportional to the mass of diformazan produced, were calculated from video-photometric measurements of the change in light intensity over individual cells. Total diformazan produced increased 78 and 126% with an increase in NBT from 0.5 to 1.0 and 2.0 mg/ml, respectively. Total diformazan produced and maximum rate of production among individual PAM varied two- to threefold providing strong evidence for heterogeneity in O₂^- production. Specific inhibition of O₂^- production by superoxide dismutase, iodoacetate, and chlorpromazine significantly reduced the total diformazan produced and maximum rate of diformazan production. Hydrogen peroxide was not involved in NBT reduction, since catalase alone did not significantly change diformazan production. This novel method to quantify O₂^- release from single PAM should be valuable in analyzing heterogeneity and single cell kinetics of O₂^- production, in assessing the effects of exposure of cells to particulates on O₂^- release, and in relating release to electrophysiological measurements.