PODEROSO Juan Jose
capítulos de libros
Kinetics of nitric oxide and reactive oxygen species production and formation of peroxynitrite by phorbol 12-myristate 13-acetate-stimulated human neutrophils. Part I. Enzymology and Biochemistry
CARRERAS, MC, CATZ, SD, PARGAMENT, GA, DEL BOSCO, CG, BOVERIS, A, PODEROSO, JJ
The Biology of Nitric Oxide, Part 4, Enzymology, Biochemistry and Immunology
Año: 1994; p. 172 - 175
There are few reports about nitric oxide (NO) production and a possible interaction with the respiratory burst in human neutrophils (PMN). To that purpose, we isolated PMN of venous samples from human healthy volunteers by Ficoll-Hypaque density gradients. NO production was measured by oxidation of oxymyoglobin (oxyMb) in a double beam-double wavelength spectrophotometer Perkin-Elmer, hydrogen peroxide (H2O2) by the fluorometric oxidation of p-hydroxyphenyl acetic acid and oxygen (O2) uptake with a Clark-type electrode. The measurements were done at different concentrations of phorbol 12-myristate 13-acetate (PMA) as stimulator and in some experiments, in the presence of 0.5-1 mM N-monomethyl L-arginine (L-NMMA) and 50-100 U/ml superoxide dismutase (SOD). To detect peroxynitrite formation, chemiluminescence of stimulated PMN (QL) was determined in an EMI photon counter with luminol. The results showed that NO production started before H2O2 was detected, at 0.1 ng/ml PMA, with a rate of 0.26±0.05, increasing up to 0.60±0.08 nmol. min-1. 106 cells-1 from 0.01 to 1 µg/ml PMA. H2O2 production started at 0.5 ng/ml PMA and increased progressively to 1.20±0.05 nmol. min-1. 106 cells-1 at 1 µg/ml PMA. PMN O2 uptake was 0.65±0.12 at 0.1 ng/ml PMA and increased up to 3.32±0.56 nmol. min-1. 106 cells-1 at 1µg/ml PMA. L-NMMA diminished NO production by 50% and also reduced O2 uptake at low PMA concentrations. Conversely, SOD increased significantly NO measurements only at high PMA levels by 30%. QL was inhibited by 50% by L-NMMA and SOD in a dose-dependent manner. We conclude that: 1. At low PMA concentrations, human PMN only produce NO and O2 uptake represents mainly NO production. 2. With higher PMA levels NO is underestimated by the oxyMb method probably by peroxynitrite formation. 3. Formation of peroxynitrite reflected by QL is inhibited either by L-NMMA or SOD.