Nongenomic Thyroxine‐Induced Relaxation of Rat Skeletal Muscle Arteries Is Mediated by Integrin αvβ3, Integrin‐Linked Kinase, ERK1/2 and ROCK

Abstract

Thyroid hormones (TH) can decrease total peripheral resistance by changing vascular tone. They induce genomic effects which are necessarily associated with the change in gene expression and nongenomic effects which could involve a wide spectrum of molecular pathways resulting in relatively rapid vasodilation (within several minutes). The mechanisms behind the nongenomic vasorelaxation are poorly understood and may differ among the vascular beds. Skeletal muscles resistance arteries are important contributors to the total peripheral resistance. Therefore, in this study, we tested the hypothesis that both TH (T3 and T4) induce rapid nongenomic dilation of rat skeletal muscle arteries. Gastrocnemius feed (sural) arteries from Wistar rats (m=300-450 g) were mounted in wire myograph to record their responses in isometric mode. We discovered that T4 (10 microM) induced more prominent relaxation than T3 (10 microM) in arteries preconstricted by methoxamine (α1-adrenoceptor agonist): to 37.7±6.6% and 67.9±11.8 % from contraction level in control, respectively. Preincubation with T4 (10 microM) was also more effective in depressing contractile responses to methoxamine than preincubation with T3 (10 microM). T3-induced relaxation disappeared after endothelium removal, while T4-induced relaxation did not depend on the presence of endothelium. Further, we studied the mechanisms of the more prominent nongenomic effect of T4 in details. The effect of T4 in endothelium-denuded arteries was abolished by tetrac (3 microM), the competitive inhibitor of integrin αvβ3. Integrin αvβ3 may mediate the extracellular matrix signalling by activation of associated kinases like Src-kinase, integrin-linked kinase (ILK) or ERK1/2. Src-kinase inhibitor PP2 (10 microM) did not affect T4-induced relaxation. However, in the presence of ILK inhibitor compound 22 (10 microM) or ERK1/2 inhibitor U0126 (10 microM), the effect of T4 was absent. One of the downstream targets of ILK could be RhoA-activated kinase (ROCK) which potentiates smooth muscle contraction by inhibiting myosin light chain phosphatase through MYPT1 phosphorylation. ROCK inhibitor Y27632 (3 microM) decreased the effect of T4. The results of functional studies were supported by Western blot data. Preincubation with T4 (10 microM) reduced the contents of phosphorylated MYPT1 (Thr850) and MLC2 (Ser19) in endothelium-denuded arterial samples, the effect of T4 was not observed in the presence of ILK inhibitor compound 22. To conclude, T3 induces endothelium-dependent rapid nongenomic relaxation of rat sural arteries. The rapid relaxing effect of T4 is more powerful and, unlike T3, endothelium-independent. The molecular pathway of T4-induced relaxation of smooth muscle includes suppression of extracellular matrix signalling via integrin αvβ3 and downstream kinases ILK, ERK 1/2 and ROCK with subsequent decrease of MYPT1 (Thr850) and MLC2 (Ser19) phosphorylation.