Nitric oxide ('NO) release, oxygen uptake and hydrogen peroxide (H202) production elicited by increasing phorbol12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 pg PMA/ml CNO) and O.OOl~.Ol pg PMA/ml (H,OJ. At saturated PMA concentrations, 'NO production, oxygen uptake and H,O, release were 0.56 ? 0.04, 3.32 f 0.52 and 1.19 f 0.17 nmol min-' 1 O6 cells-'. '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 0.00-0.01 pg PMA/ml. Preincubation with A'"-monomethyl-L-arginine (L-NMMA) decreased 0, uptake and 'NO release but increased H,Oz 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 O;-release favour the formation of other oxidants like peroxynitrite.

We studied nitrogen radical nitric oxide (.NO) release and reactive oxygen species (ROS) production by isolated neutrophils after phorbol myristate acetate (PMA) stimulation in 12 newly diagnosed and nine treated Parkinson's disease (PD) patients and 10 age-matched healthy controls. Neutrophils of both groups of PD patients had an elevated PMA-activated release of .NO [61 and 57%, respectively, higher than that of controls (p < 0.05)]. In contrast, H2O2 release was only significantly increased by 56% in chronically treated patients. In agreement, the maximum rate of luminol-dependent chemiluminescence, which partly represents O2- H2O2- .NO interactions, was increased only in the treated group. When other blood markers of oxidative stress were compared, only erythrocyte catalase activity was decreased in both PD patient series by 33 and 39%, respectively (p < 0.05), whereas plasma antioxidant capacity and erythrocyte superoxide dismutase activity levels were decreased only in treated PD patients. This study suggests that neutrophils express a primary alteration of .NO release in PD patients, whereas H2O2 and oxidative-stress parameters are more probably related to the evolution of PD or to effects of treatment with L-dopa.

The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O-2) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O-2 by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-gamma), or tumor necrosis factor (TNF-alpha) alone or in combination. Results showed that PMN release of both NO and O-2 was reduced in septic samples; in contrast, an association of LPS, IFN-gamma, and TNF-alpha promoted maximal NO release by normal cells (40-50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O-2 release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.

No abstract

Human neutrophils (PMN) activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O2.-) and its dismutation product, hydrogen peroxide (H2O2). To assess whether .NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 micrograms/ml) decreased H2O2 yield without significantly changing .NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 micrograms/ml) completely abolished H2O2 release by fMLP-activated neutrophils; conversely, .NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased .NO production and increased O2.- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H2O2 and .NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O2.- and H2O2 generation and simultaneously maintains .NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O2.- and .NO production.