Effect of NOS inhibition on mitochondrial function in Brain Microvascular endothelial cells under normoxia and oxygen‐glucose deprivation‐reoxygenation (OGD‐R)
Authors: Sure Venkata N. , Sakamuri Siva S. V. P. , Evans Wesley R. , Sperling Jared A. , Peterson Nicholas R. , Chen Allen L. , Zheng Sufen , Katakam Prasad V. G. 
Published: June 2021

Objective

Brain Microvascular endothelial cells (BMECs) form the blood‐brain barrier (BBB) with other cell types such as microglia, astrocytes end feet, pericytes, and a basement membrane. Ischemic stroke damages BBB causing brain injury during reperfusion. Ischemic stroke in endothelial nitric oxide synthase (eNOS) knockout mice showed increased infarct size compared to wild‐type and the underlying mechanisms are not clear. eNOS produces nitric oxide which plays an important role in maintaining vascular homeostasis. Mitochondria play a key role in pathophysiological conditions such as ischemic stroke and diabetes. In addition, NOS has been showed to uncouple and produce superoxide during pathological conditions. Our objective is to study the effect of NOS inhibition on mitochondrial function in BMECs under normoxic and OGD‐R conditions.

Methods

BMECs isolated from 2 weeks old Sprague Dawley rat pups were exposed to normoxic and OGD‐R conditions with and without NOS inhibitor N‐ω‐propyl‐L‐arginine (L‐NAME). We used Seahorse XFe 24 bioanalyzer (Agilent technologies) to measure mitochondrial oxygen consumption rates (OCR) using mito‐stress assay. Reactive oxygen species (ROS) levels were measured using Electron spin resonance (ESR) spectrometry (Bruker Bio‐Spin spectrometer, Germany). In addition, mitochondrial membrane potential was measured using Rhodamine 123 fluorescence and CCK‐8 kit was used to measure cell viability.

Results

NOS inhibition using L‐NAME in BMECs under normoxic conditions reduced basal and maximal respiration. However, there is no significant difference in spare respiratory capacity, proton leak and non‐mitochondrial respiration in L‐NAME treated cells compared to the control group under normoxic conditions. Interestingly, NOS inhibition in BMECs exposed to OGD‐R decreased all the parameters of mitochondrial respiration such as basal respiration, maximal respiration, proton leak, and spare respiratory capacity compared to control group. ESR measurements using ROS specific spin probe CMH showed that NOS inhibition reduced ROS production compared to untreated cells in BMECs exposed to both normoxic and OGD‐R conditions. Fluorescence measurements using Rhodamine 123 have shown that cells exposed to OGD‐R reduced mitochondrial membrane potential and L‐NAME treatment improved mitochondrial membrane potential whereas, in cells grown under normoxic conditions, NOS inhibition has no significant effect on mitochondrial membrane potential. Finally, viability studies have shown that OGD‐R decreases cell viability and NOS inhibition in BMECS exposed to OGD‐R improves cell survival whereas, in normoxic conditions, L‐NAME treatment reduced cell viability after 24 hours.

Conclusion

NOS inhibition using L‐NAME in BMECs exposed to OGD‐R improves cell survival rate by decreasing ROS production and by reducing mitochondrial respiration.

Support or Funding Information

National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.