Paradoxical mitochondrial effects of nitric oxide synthase inhibition following oxygen-glucose deprivation-reoxygenation (OGD/R) in endothelial cells and neurons.

Current dogma in stroke is that neuronal nitric oxide synthase (NOS1) exacerbates ischaemic brain injury, while endothelial nitric oxide synthase (NOS3) is protective. However, oxidative stress from ischaemia-reperfusion and oxygen-glucose deprivation-reoxygenation (OGD/R) is known to uncouple NOS, leading to increased production of reactive oxygen species (ROS). This study investigated whether the inhibition of NOS uncoupling in rat brain microvascular endothelial cells (BMECs) and neurons is cytoprotective against OGD/R-induced injury. All experiments were conducted in both BMECs and neurons under normoxic conditions and following OGD/R, with or without NOS inhibition. Cell viability was assessed using Cell Counting Kit-8. Electron spin resonance spectrometry measured ROS and NO production, while mitochondrial membrane potential (MMP) was evaluated using rhodamine 123 fluorescence. Oxygen consumption rate (OCR) was measured to assess mitochondrial respiration. NOS inhibition improved post-OGD/R survival in BMECs and neurons accompanied by a reduction in NOS-derived ROS. Interestingly, while BMECs showed both ROS-producing uncoupled NOS and NO-producing coupled NOS, neurons showed NO-producing NOS only. Under normoxic conditions, NOS inhibition reduced mitochondrial respiration in BMECs but increased OCR in neurons. OGD/R led to impaired mitochondrial respiration in both BMECs and neurons, with further reductions observed following NOS inhibition. NOS inhibition in BMECs and neurons elicit distinct mitochondrial effects under normoxia but promotes identical paradoxical suppression of mitochondrial respiration in response to OGD/R. NOS uncoupling instigates post-OGD/R cellular injury in both BMECs and neurons and is a potential therapeutic target in stroke.