Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria

Abstract

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (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 nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. 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 effect 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 the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms. NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

Glutathione in Protein Redox Modulation through S-Glutathionylation and S-Nitrosylation

“…In addition, the isoform mtNOS, a homolog of NOS1, has been found in the inner mitochondrial membrane and matrix. Its influence on the mitochondrial function has been intensively studied in recent years [ 72 , 73 ].…”

The Relationship between Myoglobin, Aerobic Capacity, Nitric Oxide Synthase Activity and Mitochondrial Function in Fish Hearts

“…No significant relationships could be established between NOS activity measured in the mitochondrial suspensions and mitochondrial NO IC 50 or ventricular NOS activity (Figure 5E,F). The existence of mitochondrial NOS (mtNOS), identified as a nNOS by most studies, and its functional role in regulating mitochondrial function is still being debated [74][75][76][77]. Mitochondrial NOS activity in our Hb + Mb + fish (0.15-0.10 nmol mg protein −1 min −1 ) is slightly lower than that measured in porcine heart mitochondria (0.22 nmol mg protein −1 min −1 ; [78]).…”

Valosin-containing protein acts as a target and mediator of S-nitrosylation in the heart through distinct mechanisms

“…SNO can also modulate the activity of ion channels in cardiac myocytes, such as potassium channels and L-type calcium channels, to maintain normal cardiac rhythm and function [ 36 , 37 ]. In addition, SNO can modulate mitochondrial function in cardiac cells by affecting the activity of proteins involved in mitochondrial respiration, ATP production, and apoptosis [ 38 , 39 ]. Importantly, SNO has been shown to confer protection against ischemia-reperfusion injury by modulating various signaling pathways involved in cell survival and apoptosis [ 9 , [40] , [41] , [42] ].…”