The assay was carried out according to the instructions of the manufacturer

eading to an increased vascular relaxation. AT2R-mediated vasodilatation has been reported in both endothelium-dependent and independent manners. Based on AT2R upregulation observed in cardiomyocytes from the hearts of rats induced to hyperthyroidism we evaluated the possible involvement of this receptor in VSMC with increased vascular relaxation observed in this pathology. We observed increased levels of AT2R in the aortas from hyperthyroid animals, corroborating with our initial hypothesis that AT2R might be involved in vascular relaxation found in hyperthyroidism. In addition, AngI/AngII protein expression levels were also significantly increased in aortas from hyperthyroid animals, indicating that T3 also increased the levels of the ligand AngII. The action of TH increasing local Ang II levels has already been described in other cell types. We also assessed the direct effect of T3 on cultured VSMC in order to investigate the possible molecular mechanisms involved in hormone action on relaxation response. According to the observations in endothelium-denuded aortas, AT2R was present under basal conditions in VSMC and its mRNA 22022974 and protein expression levels were upregulated after T3 treatment with no changes on AT1R levels. Similarly, treatment of VSMC with T3 promoted a reduction in p-MLC levels in parallel with a decrease on alpha-actin levels, confirming the effect of T3 on reducing contractile proteins. This effect of TH on the contractile protein levels in VSMC has not been demonstrated previously in the literature. Taking into account recent evidence 7623957 showing that AT2R stimulation could act on the contractile machinery modulating processes such as depolymerization of actin, we tested the hypothesis of a possible correlation between upregulation of AT2R and decreased contractile proteins found in VSMC stimulated with T3. Interestingly, VSMC treated previously with AT2R blocker partially reversed the effect of T3, showing for the first time an involvement of this receptor in the action of T3 in the reduction of contractile proteins, possibly to increase the vascular relaxation found in the hyperthyroidism. Based on results obtained in vitro we performed functional studies using vascular reactivity experiments in order to evaluate the role of AT2R on relaxation induced by T3. Endotheliumdenuded aorta rings from hyperthyroid animals presented a lower response to Ang II, compared to control. This effect was partially reversed when the rings were pre-incubated with PD123319, indicating that the lower contractile response occurs at least in part to the involvement of AT2R. In fact, previous studies demonstrated that AT2R AMI-1 located in smooth muscle may directly mediate vasodilatation effects, but also may minimize the contraction promoted by Ang II. Besides the morphological arrangement and all the vessel contractile components, aorta is responsive to numerous vascular modulators including TH, which makes it AT2R Mediates T3-Induced VSMC Relaxation a good model to study vascular contractile or relaxation phenomena. In addition, this vessel serves as an important tool to obtain primary cultures of VSMC. One of the mechanisms by which AT2R promotes vasodilatation is through nitric oxide production , which seems to occur after stimulation of bradykinin by AT2R. Thus, an assay to indirectly evaluate the NO levels through the nitrite concentrations determination was performed in cultured VSMC submitted to T3 treatment in the presence or absence o