als during treatment, while T3 induced a significant increase after 14 days of treatment. Taken together these results confirm the efficiency of T3 treatment inducing hyperthyroidism in these rats. T3-treatment Decreases Contraction to Ang II in the Aorta via AT2R In order to determine whether the upregulation of AT2R contributes to HC-030031 cost T3-induced relaxation, concentration-response curves to angiotensin II were performed in endothelium-denuded aortic rings from Hyper and control rats, in the presence or absence of PD123319, a specific AT2R blocker. Ang II produced concentration-dependent contraction in all tested aortic rings. Aortas from Hyper rats displayed decreased contraction to Ang II compared with control aortas and this effect was attenuated in the aortas pre-incubated with PD123319 . No differences were observed in the pD2 among the groups. These data suggest AT2R is involved in decreased contraction to AngII in hyperthyroid rats. T3-treatment Decreases Contractile Protein Levels in Aortas One likely explanation for the vascular relaxation present in hyperthyroidism is a reduction in proteins related to smooth muscle contraction. Indeed, phosphorylated MLC levels, a 6145492 well-known marker of contraction, were decreased by 17526600 30% in T3 Increases AT2R Gene and Protein Expression Levels in Cultured VSMC Based on the effects of hyperthyroidism on AT2R expression in aortas, we examined the effect of T3 treatment in cultured VSMC. The AT2R mRNA levels were significantly increased in a T3 concentration-dependent manner . Further, VSMC stimulated with T3 for 24 AT2R Mediates T3-Induced VSMC Relaxation 4 AT2R Mediates T3-Induced VSMC Relaxation hours showed augmented AT2R and unchanged AT1R protein expression, which was accompanied by increased AngI/II levels. These results indicate that T3 plays important role in local activation of RAS. AT2R Mediates the T3-induced Diminution of Contractile Protein Expression in Cultured VSMC In order to investigate whether AT2R contributes to T3induced vascular relaxation, cells were treated for 24 hours with T3 and/or pre-incubated with AT2R blocker. T3 induced a significant decrease in p-MLC and a-actin levels in cultured VSMC, which was partially reversed in cells preincubated with PD123319 for 30 min. These data support the previous results found in aortas from T3-treated rats and also show that the direct effect of T3 in diminishing the contractile apparatus proteins is mediated by AT2R. T3-induced NO Production Occurs via AT2R/Akt Pathway We previously reported that T3 rapidly stimulates NO production in VSMC. Using nitrite levels assay, we observed that the levels of NO22 were augmented by 2 fold in supernatants of cultured VSMC stimulated with T3. This effect was diminished in cells pre-incubated with PD123319. Because the PI3K/Akt signaling pathway is downstream of AT2R and is involved in the vascular NO production, 
we next examined the effects of wortmannin, a selective inhibitor of PI3K, in T3-induced NO production. The increased levels of NO in VSMC stimulated with T3 were partially reversed in the presence of wortmannin. To confirm whether T3induced PI3K/Akt activation occurs via AT2R, we measured activation of PI3K/Akt by phosphorylation in the presence of PD123319. We observed increased levels of phosphorylation Akt at Ser473 and Thr308 in VSMC stimulated with T3; however, in the presence of PD123319, this effect was reduced, showing that T3 activates PI3k/Akt signaling via AT2R. Together these re