Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Foundation for the Support of Research in the State of Sao Paulo; grants 06/61523-7 and 06/54064-6)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Foundation for the Support of Research in the State of Sao Paul

Effect of Hyperthyroidism (Hyper) in the protein expression.

<p>Hyper reduced the expression of contractile proteins: p-MLC (A), α-actin and calponin (B). In addition, Hyper was associated with increased AT2R (C) and AngI/II (D) levels in aortas. Top: representative blots. Bottom: Densitometry values. Data are expressed as mean ± SD (n = 6) per group, *p<0.05 <i>vs.</i> control.</p

AT2R mediates endothelium-independent decreased contraction in aortas from hyperthyroid rats.

<p>Contractile response to AngII (from 0.1 nM to 0.1 µM) is decreased in hyper denuded-aortas (open square) when compared to control group (open circle). The specific blockade of AT2R with 0.1 µM PD123319 (filled square) reverses this effect when compared to control group pretreated with PD (filled circle). Data are represented as mean ± SEM (n = 4 per group, *p<0.05 <i>vs.</i> control group, #p<0.05 <i>vs.</i> control+PD).</p

Schematic representation of the proposed model.

<p>T3 induces a sustained increase in AT2R and AngI/II levels, which in turn causes the activation of PI3K pathway with the consequent increment in Akt phosphorylation that finally leads to an augmentation in NO production. Besides, T3 also causes a diminution in contractile protein levels. Altogether, these effects promote an increase in vascular relaxation. On the other hand, the specific blockade of AT2R (with PD123319) is able to partially or completely abolish the effects of T3, culminating in the prevention of the increase of vascular relaxation and evidencing the role of AT2R/PI3K/Akt pathway in these processes.</p

AT2R mediates T3-induced PI3K/Akt activation.

<p>Cultured VSMC stimulated with T3 (0.1 µM for 24 hours) display increased levels of p-Akt in both residues (thr308 and ser473), which is partially reversed by the specific blockade of AT2R with PD123319 (0.1 µM for 30 min prior to T3 treatment). Data are expressed as mean ± SD (n = 6 per group, *p<0.05 <i>vs.</i> control, #p<0.05 <i>vs.</i> T3).</p

AT2R blocker reduces T3-induced decreased levels of contractile proteins and attenuates T3-induced NO production.

<p>Decreased levels of p-MLC (A) and α-actin (B) in VSMC treated with T3 (0.1 µM) for 24 h were attenuated in the VSMC pre-incubated with PD123319 (0.1 µM) for 30 min. Pre-incubation with PD123319 (0.1 µM) (C) and wortmannin (0.1 µM) (D) attenuates the T3-induced augmented NO generation in VSMC. In (C), NO₂⁻ concentration was measured by chemiluminescence, while in (D) NO production was determined was determined using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2). Data are expressed as mean ± SD (n = 5 per group for A and B; n = 4 per group for C and D; *p<0.05 <i>vs.</i> control, #p<0.05 <i>vs.</i> T3, $p<0.05 vs. PD).</p