| Summary: | Myoendothelial junctions (MEJs) are structures that allow chemical signals to be transmitted between endothelial cells (ECs) and vascular smooth muscle cells, which control vascular tone. MEJs contain hemoglobin alpha (Hbα) and endothelial nitric oxide synthase (eNOS) complexes that appear to control the production and scavenging of nitric oxide (NO) along with the activity of cytochrome b5 reductase 3 (CYB5R3). The aim of this study was to examine how hypoxia affected the regulation of proteins involved in the production of NO in brain ECs. In brief, human brain microvascular endothelial cells (HBMEC) were exposed to cobalt chloride (CoCl<sub>2</sub>), a hypoxia mimetic, and a transcriptional analysis was performed using primers for <i>eNOS</i>, <i>CYB5R3</i>, and <i>Hbα2</i> with ΔΔCt relative gene expression normalized to <i>GAPDH</i>. NO production was also measured after treatment using 4,5-diaminofluorescein diacetate (DAF-DA), a fluorescent NO indicator. When HBMEC were exposed to CoCl<sub>2</sub> for 48 h, <i>eNOS</i> and <i>CYB5R3</i> messenger RNA significantly decreased (up to −17.8 ± 4.30-fold and −10.4 ± 2.8, respectively) while <i>Hbα2</i> increased to detectable levels. Furthermore, CoCl<sub>2</sub> treatment caused a redistribution of peripheral membrane-generated NO production to a perinuclear region. To the best of our knowledge, this is the first time this axis has been studied in brain ECs and these findings imply that hypoxia may cause dysregulation of proteins that regulate NO production in brain MEJs.
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