Activation of Endothelial Nitric Oxide Production by the Sigma Receptor Agonist Afobazole

Abstract

Afobazole is a sigma‐1 receptor agonist that has been shown to enhance survival in a rat mid‐cerebral artery occlusion stroke model. The mechanism has been previously attributed to an inhibition of ischemia‐induced calcium dysregulation that lowers glial cell loss and infarct volume. A recently published finding is that afobazole can stimulate nitric oxide (NO) production from lymphatic endothelial cells. In the current study, we investigated the ability of afobazole to relax arterioles from the brain and potential interactions between the sigma‐1 receptor and endothelial NO synthase (eNOS). Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. Afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM. The ability of afobazole to elicit NO production from cultured human brain microvascular endothelial cells (HBMEC) was assessed using 4‐Amino‐5‐Methylamino‐2′,7′‐Difluorofluorescein Diacetate (DAF‐FM) loading and fluorescence microscopy. Phosphorylation of eNOS on Ser‐1177 in the absence or presence of afobazole was evaluated by Western blotting. Lastly, intracellular localization of eNOS and the sigma‐1 receptor were evaluated by immunofluorescence and laser confocal microscopy. The results show that 200 and 250 μM afobazole elicited significant dilation of parenchymal arterioles. Afobazole also elicited elevated NO production from HBMEC in association with elevated phosphorylation of eNOS on Ser‐177. Lastly, eNOS and the sigma‐1 receptor were found partially colocalized in what appears to be the Golgi complex in confocal image stacks of HBMEC. This study shows for the first time a novel mechanism by which afobazole can relax brain arterioles via eNOS‐mediated NO production. This mechanism is likely associated with previously reported improvements of outcomes following stroke induced brain injury.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.