Transcriptional and translational mechanisms for the reciprocal control of iNOS and endothelin 1 expression in brain microvessels after traumatic brain injury (TBI)

Abstract

Previously we demonstrated in a rat model (Marmarou's) that in vivo inhibition of iNOS gene expression (not observed in the intact brain and a source of abnormally high levels of nitric oxide (NO)) results in upregulation of the endothelin 1 (ET-1) gene and protein expression at the microvascular wall1. We attributed the exacerbated hypoperfusion and enhanced vasoconstriction to increased synthesis and release of ET-1, a powerful vasoconstrictor. In addition, we have shown that iNOS and ET-1 are synthesized in the same endothelial cells that form the microvascular wall2. Recently we observed, post TBI, induction of c-fos expression (the immediate early gene whose expression is essential for the activation of the AP-1 cascade and the ET-1 gene). Also, we showed and increased synthesis of the phosphorylated form of eukaryotic initiation factor 2 alpha (eIF2aP phosphorylation of the a subunit of causes inhibitor of translation of protein synthesis)3. Here we test the hypothesis that c-fos and eIF2aP are temporally associated with the enhanced expression of ET-1 after TBI and that iNOS expression and inhibition plays a role in regulating their synthesis. We used combinations of double immunocytochemistry and in situ hybridization to detect expression of ET-1, iNOS, c-fos and eIF2aP in cortex and hyppocampus of male Sprague-Dawley rats at 4, 24 and 48 h post TBI. Other animals were pretreated with antisense iNOS oligodeoxynucleotides (ODNs) to block the mRNA synthesis. In the latter group expression of c-fos was increased in ET-1 positive cells (endothelial cells, pericytes, neurons and astroglia) by 25% in comparison to brains from animals that were not pretreated with antisense ODNs. In the same group, the number of cells that coexpressed iNOS and eIF2aP was reduced by 32% and the intensity of eIF2aP immunoreactivity decreased by 38%.The effectiveness of the iNOS knockout was confirmed with in situ hybridization and Western blotting. The results suggest that further upregulation of ET-1 synthesis in animals subjected to TBI and pretreated with antisense iNOS ODNs is related to: 1) disinhibition of AP-1 since NO is known to suppress its expression at several levels including c-fos and c-jun, the protein product of the latter forming a dimer with c-fos (the protein) that binds to AP-1 and, 2) by reducing the amount of eIF2aP since previous in vitro work has shown, that NO is a phosphorylating molecule. Therefore, by decreasing the synthesis of NO we may have disinhibited protein synthesis including ET-1. In addition, our finding that c-fos is localized in the nucleus, while eIF2aP is observed in both nucleus and cytoplasm, suggests that NO can influence their synthesis at the transcriptional and translational level.