P-169 - Macrophage-derived peroxynitrite at physiological O2 concentration

Abstract

Cell cultures are performed at 20% O2 (95% air/5% CO2) while a range of pO2 between 2–5% is found in different tissues (20–50 µM O2 at 37 °C, pH7.4). Peroxynitrite formation may be affected by pO2 since the sources of superoxide radical (O2•−) and nitric oxide (•NO) are enzymes that utilize oxygen as substrate. Neutrophils/macrophages contain NADPH oxidase (NOX-2), activated during phagocytosis, generating O2•– in a “respiratory burst” that consumes O2 at rates greater than basal levels. The reported Km for neutrophil NOX-2 is 2–3% O2, so theoretically, it would keep 45–70% of its maximum activity at physiological pO2 (2–5%). Nitric oxide (•NO) derives from the inducible nitric oxide synthase, with a higher Km (~11% O2) being more sensitive to changes in oxygen concentration in the physiological range. Experimental results in macrophages show that O2•− generation at 2% is 64% of that obtained in cells incubated at 20% O2. Despite what was expected, •NO production was similar for both conditions. Peroxynitrite formation is being assessed using fluorescein-boronate under different oxygen tensions. The quantitative data of •NO, O2•− and peroxynitrite fluxes under physiological O2 will provide a more accurate insight into the biological interplay and role of •NO-derived oxidants.