Slow exponential decay of rate of superoxide production in phorbol ester-activated human neutrophils

Proper quantification of the superoxide (O2-) respiratory burst induced in human neutrophils is important for better understanding of the mechanism of action of stimulators and inhibitors. Reexamination of the reaction triggered by phorbol myristate acetate (PMA) indicated that it was a persistent process which lasted over 60 min. Plots of rates versus time show that rates of O2- release decayed logarithmically with a mean half-life (T1/2) of 21 +/- 6 min (SD), N = 12). Calculations of areas under curves indicate an average O2- yield of 217 +/- 99 nmol/10(6) cells. The inclusion of catalase in incubation mixtures did not alter the T1/2 or O2- yield, nor was the latter value affected by the quantitive scavenging of O2- by cytochrome c. Under certain conditions--the presence of excess dimethyl sulfoxide, the substitution of a less potent phorbol ester or activation of cells at high densities--the initial rate was either increased or decreased but a complementary alteration in the T1/2 resulted in little or no change in the total O2- yield. Retinol and retinol acetate decreased the initial rate, but retinoic acid enhanced it. By comparison, total O2- production was markedly reduced by all three agents with the following order of potency: retinoic acid greater than retinol greater than retinol acetate. In contrast, the serine protease inhibitor, TPCK, suppressed both the O2- yield and initial rate to a similar extent. On the basis of present observations, it is proposed that under normal conditions of PMA cellular activation, the logarithmic decay of the rate of O2- release was not due to autoinactivation of the O2--generating system, but rather to another factor, a possibility being the depletion of intracellular NADPH.