The Effects of Nitric Oxide Synthase Inhibitors on Mitochondrial Respiration in Isolated Mouse Brain Mitochondria

Abstract

ObjectiveNitric oxide synthase (NOS) plays an important role in intracellular and intercellular signaling pathways. Experimental studies in mitochondria from various tissues, including the brain, have shown the possibility of the presence of a novel mitochondrial variant of NOS (mtNOS) located within the mitochondrial matrix. We hypothesized that administration of NOS inhibitors significantly affects the mitochondrial respiration in isolated brain mitochondria.MethodsMitochondria were isolated from the mice aged 8–12 weeks old and plated in 24‐well microplates for oxygen consumption rate (OCR) readings in the Seahorse XFe24 Analyzer. Mitochondria were incubated with inhibitors of nNOS (ARL‐17477 and N‐ω‐propyl‐L‐arginine), eNOS (N5‐(1‐iminoethyl)‐L‐ornithine), or a non‐specific NOS inhibitor (L‐NG‐Nitroarginine methyl ester) for 30 minutes prior to the mitochondrial stress test in the XFe24 Analyzer and compared to an untreated control group.ResultsPharmacological inhibition of nNOS in isolated brain mitochondria reduced basal respiration and ATP production. Treatment with nNOS‐specific and non‐specific NOS inhibitors showed reduced basal OCR compared to control mitochondria. Control OCR values of 90.76±1.89 pmol/min were significantly higher than treatment with L‐NAME (74.17±2.12 pmol/min), NPA (77.92±2.83 pmol/min) and ARL (76.60±2.85 pmol/min). eNOS‐specific inhibitor treatment showed no significant change in mitochondrial respiration. ATP production as a bioenergetics parameter represents the portion of basal oxygen consumption that was used to drive ATP synthesis. Isolated mitochondria treated with non‐specific inhibitor L‐NAME and nNOS‐specific ARL‐17477 showed a significant decrease in ATP production. Isolated mitochondria treated with NPA show a slight trend towards decreased ATP production, but the statistical analysis did not show significance. Treatment with NIO demonstrated no significant change in ATP production.ConclusionsPharmacological inhibition of NOS isoforms affects brain mitochondrial respiration. Given the significant effects demonstrated by both nNOS‐specific inhibitors and L‐NAME, it may be postulated that mtNOS may be a new isoform of NOS sharing specific properties of the traditional three isoforms.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006); National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834); and National Institute of Diabetes and Digestive and Kidney Diseases (RS: DK107694).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.