Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NOS in the mitochondria (mtNOS) of cells, however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear.
Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain non-synaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1µmol/L) or endothelial (eNOS; N5-(1-iminoethyl)-L-ornithine, NIO, 1µmol/L) NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates (OCR) measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and western blotting. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting.
nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no affect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing inhibitory role attributed to NOS isoforms.
