Diaphragmatic nitric oxide synthase is not induced during mechanical ventilation

Mechanical ventilation (MV) is associated with diaphragmatic oxidative stress that contributes to both diaphragmatic atrophy and contractile dysfunction. However, the pathways responsible for oxidant production in the diaphragm during MV remain unknown. To address this issue, we tested the hypothesis that diaphragmatic nitric oxide synthase (NOS) activity is elevated during MV, resulting in nitration of diaphragmatic proteins. Rats were mechanically ventilated for 18 h, and time-matched, anesthetized but spontaneously breathing animals served as controls. Protein levels of endothelial NOS, inducible NOS, and neuronal NOS were measured in diaphragms from all animals. 3-Nitrotyrosine levels were also measured as an index of protein nitration, and S-nitrosothiol levels were measured as a marker of nitric oxide reactions with molecules containing sulfhydryl groups. Levels of nitrates and nitrites were measured as markers of stable end products of nitric oxide metabolism. Finally, as a marker of oxidative stress, diaphragmatic levels of reduced GSH were also analyzed. MV did not promote an increase in diaphragmatic protein levels of endothelial NOS or neuronal NOS. Moreover, inducible NOS was not detected in the diaphragms of either experimental group. Consistent with these findings, MV did not elevate diaphragmatic 3-nitrotyrosine levels in any subcellular fraction of the diaphragm, including the cytosolic, mitochondrial, membrane, and insoluble protein fractions. Moreover, prolonged MV did not elevate diaphragmatic levels of S-nitrosothiols, nitrate, or nitrite. Finally, prolonged MV significantly reduced diaphragmatic levels of GSH, which is consistent with diaphragmatic oxidative stress. Collectively, these data reveal that MV-induced oxidative stress in the diaphragm is not due to increases in nitric oxide production by NOS.