Aim: 

We have evaluated the temporal contribution of nitric oxide in the inhibition of respiration in sepsis and the influence of concurrent hypoxia.

Methods: 

Human umbilical endothelial cells (HUVECs) were activated by endotoxin, TNFalpha and IFNgamma over a 24-h period. Thereafter cells were incubated at 21% and 1.5% oxygen (normoxia and hypoxia respectively) and mitochondrial respiration, complex I activity, peroxynitrite production, S-nitrosylation, antioxidant content and tyrosine nitration were evaluated by oxygen Clark electrode, fluorescent probes and western blot.

Results: 

Oxygen consumption, complex I activity and antioxidant levels fell progressivelly over time in activated cells. This was largely prevented by coincubation with the non specific inhibitor N-nitro L arginine. Addition of glutathione ester and mitoquinone, two mitochondrial antioxidants, reversed these effects an initial time points. Thereafter, the inhibition of complex I became more persistent, coinciding with a progressive increase in nitration. Hypoxia accelerated the persistent inhibition of complex I.

Conclusion: 

Our results suggest that hypoxia amplifies the mitochondrial inhibition induced by NO generated during inflammatory disease states such as hypoxia and show that mitochondria-targeted antioxidants as a therapeutic tool in these pathologies.