Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils

Abstract

Nitric oxide (^.NO) release, oxygen uptake and hydrogen peroxide (H₂O₂) production elicited by increasing phorbol 12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001–0.001 μg (^.NO) and 0.001-0.01 μg (H₂O₂). At saturated PMA concentrations, ^.NO production, oxygen uptake and H₂O₂ release were 0.56 ± 0.04, 3.32 ± 0.52 and 1.19±0.17 nmol · min⁻¹ · 10⁶ cells⁻¹. ^.NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.001-0.01 μg . Preincubation with N ^G-monomethyl-l-arginine (l-NMMA) decreased O₂ uptake and ^.NO release but increased H₂O₂ production, while superoxide dismutase (SOD) increased ^.NO detection by 30%. Chemiluminescence was also reduced by preincubation with l-NMMA and/or SOD. The results indicate that ^.NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of ″NO and O₂ ^.− release favour the formation of other oxidants like peroxynitrite.