Diverse effects of NOS inhibition on mitochondrial bioenergetics in rat primary cortical neuronal cells under normoxia and hypoxia

Abstract

ObjectiveIschemic brain injury has been shown to be increased in endothelial nitric oxide synthase (eNOS) knockout mice but reduced in neuronal nitric oxide synthase (nNOS) knockout mice compared to the wild‐type mice. It has been proposed that NO derived from nNOS in neurons is detrimental whereas NO derived from eNOS in endothelial cells is protective after stroke. However, the functional mechanism underlying this protection or damage has never been studied. Mitochondria play an important role in a wide range of physiological and pathophysiological processes. Besides the generation of cellular energy, mitochondria are also involved in reactive oxygen species (ROS) production and the activation of cell death pathways. Therefore, the effect of NOS inhibitors on mitochondrial respiration is important to investigate as mitochondria determine the fate of cell survival after stroke or ischemic injury. Our objective is to determine the effects of NOS inhibition on mitochondrial bioenergetics in primary rat cortical neurons under normoxic and oxygen‐glucose deprivation‐reoxygenation (OGD‐R) conditions.MethodsCortical neurons were isolated from brain and exposed to normoxic and OGD‐R conditions with and without non‐specific NOS inhibitor N‐ω‐propyl‐L‐arginine (L‐NAME). Oxygen consumption rates (OCR) and respiratory parameters were measured using Seahorse XFe24 cell mito‐stress test (Agilent technologies). Cell viability was measured using CCK‐8 kit to determine the effect of NOS inhibition under hypoxic and normoxic conditions. Electron spin resonance (ESR) spectroscopy (Bruker Bio‐Spin spectrometer, Germany) was used to measure ROS using spin probe CMH.ResultsIn neurons, OCR showed a decrease in basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition compared to normoxic control. Interestingly, NOS inhibition under normoxic conditions increased basal respiration, maximal respiration, spare respiratory capacity, and non‐mitochondrial respiration but not proton leak whereas, NOS inhibition decreased basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition. Thus, NOS inhibition has opposite effects on mitochondrial respiratory parameters under normoxic and hypoxic conditions. Viability studies showed NOS inhibition has improved cell viability under hypoxic conditions. In contrast, NOS inhibition under normoxic conditions showed a decrease in cell viability. ROS measurements by ESR have shown that L‐NAME treatment increased ROS levels under normoxic conditions, however, NOS inhibition decreased ROS levels under hypoxic conditions.ConclusionsNOS inhibition has opposite effects on mitochondrial bioenergetics, viability and superoxide levels under hypoxia compared to normoxia. nNOS appears to inhibit ROS generation under hypoxia but promotes the ROS generation following hypoxia possibly due to uncoupling of nNOS. Thus, the change in the generation of free radicals may underlie the diverse effects of nNOS in neurons following exposure to normoxia and hypoxiaSupport or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.