Diaphragmatic nitric oxide synthase is not induced during mechanical ventilation

Abstract

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 hours and time matched, anesthetized, but spontaneously breathing animals served as controls. Diaphragmatic NOS activity, protein levels of eNOS, iNOS, and nNOS were measured and 3-nitrotyrosine levels were also determined as an index of protein nitration. MV did not promote an increase in diaphragmatic protein levels of eNOS, nNOS or total NOS activity. Moreover, iNOS was not detected in the diaphragms of either experimental group. Consistent with these findings, MV did not elevate diaphragmatic 3-nitrotyrosine levels in any sub-cellular fraction of the diaphragm including the cytosolic, mitochondrial, membrane, and insoluble protein fractions. Collectively, these data indicate that MV-induced oxidative stress in the diaphragm is not due to increases in nitric oxide production by NOS. This work supported by NIH RO1 HL072789 (S.K.P) and NIH T32 HD043730 (D.J.F).