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Effects of respiratory burst inhibitors on nitric oxide production by human neutrophils
M C Carreras1, N A Riobó, G A Pargament
1University Hospital, School of Medicine, University of Buenos Aires, Argentina.
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.
Insights
Human neutrophils release nitric oxide (.NO), superoxide anion (O2.-), and hydrogen peroxide (H2O2) upon activation. Inhibitor studies reveal distinct pathways regulate these reactive oxygen species, with tyrosine kinase activating O2.-/H2O2 and protein kinase C modulating both O2.- and .NO.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Human neutrophils (PMN) are key immune cells involved in pathogen defense.
- Upon activation, PMN release reactive oxygen species (ROS) including nitric oxide (.NO), superoxide anion (O2.-), and hydrogen peroxide (H2O2).
- The signaling pathways governing the production of these ROS are complex and interconnected.
Purpose of the Study:
- To investigate the intracellular signaling pathways regulating nitric oxide (.NO) and NADPH oxidase activation in human neutrophils.
- To determine if .NO production shares common regulatory steps with superoxide anion (O2.-) and hydrogen peroxide (H2O2) generation.
- To elucidate the roles of tyrosine kinases and protein kinase C in modulating ROS production.
Main Methods:
- Human neutrophils were stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP) or phorbol ester (PMA).
- Cells were treated with specific inhibitors targeting G-proteins (pertussis toxin), tyrosine kinases (genistein), phosphotyrosine phosphatases (orthovanadate), and protein kinase C (staurosporine).
- Production of .NO, O2.-, and H2O2 was measured under various inhibition conditions.
Main Results:
- Pertussis toxin inhibited H2O2 but not .NO production in fMLP-stimulated neutrophils.
- Genistein abolished H2O2 release while increasing .NO production in fMLP and PMA-stimulated cells.
- Orthovanadate decreased .NO and increased O2.-. Staurosporine and burst antagonists diminished both H2O2 and .NO production.
Conclusions:
- Activation of the tyrosine kinase pathway positively regulates O2.- and H2O2 generation and maintains low levels of .NO in stimulated neutrophils.
- Protein kinase C activation is a positive modulator for both O2.- and .NO production.
- Distinct signaling pathways differentially regulate the production of ROS in human neutrophils.