PODEROSO Juan Jose
capítulos de libros
Nitric oxide mediates human neutrophil apoptosis
RIOBÓ, NA, PODEROSO, JJ, CARRERAS, MC
The Biology of Nitric Oxide, Part 6
Año: 1998; p. 34 - 34
Neutrophils are terminally differentiated cells that undergo spontaneous apoptosis. At resting conditions, these cells release 70±10 pmoles NO min-1 but lack any detectable production of O2- and H2O2, probably due to the formation of ONOO-. To discriminate possible effects of NO, O2-, or their reaction product peroxynitrite on spontaneous PMN apoptosis, we evaluated the viability by Trypan blue extrusion, quantitated the DNA fragmentation with the diphenylamine colorimetric assay and observed the appearance of the characteristic DNA laddering in agarose gels at 3, 6, 12, 18, and 24 h in the presence or absence of 1-5 mM L-NMMA, 1,66-5 µM SOD, 0,33-1,5 µM catalase and 0,1-1-2,5 mM GSNO. Without any addition, the spontaneous apoptotic process began at 3 h as evidenced by a very slight ladder pattern and increased over time until 24 h, when 90% of cells were still viable and 40% of total DNA was fragmented. The effects of L-NMMA were consistent with a dose-dependent inhibition of apoptosis at any studied period. Addition of GSNO clearly increased apoptosis at earliest stages but its effects were shaded over time. Catalase diminished apoptosis in a dose-dependent manner, probably by the reaction of its heme group with NO, while SOD had a dual response, enhancing it at 1,66 µM and preventing it at highest concentrations. These results point NO as a key mediator of human neutrophil apoptosis, while the role of oxygen active species and peroxynitrite seems to be the modulation of NO availability.