Angiotensin II regulates the LARG/RhoA/MYPT1 axis in rat vascular smooth muscle in vitro
Abstract
Aim: To identify a key protein that binds monomeric G protein RhoA and activates the RhoA/Rho kinase/MYPT1 axis in vascular smooth muscle cells (VSMCs) upon angiotensin II (Ang II) stimulation.
Methods: Primary cultured VSMCs from Sprague-Dawley rats were transfected with siRNAs against leukemia-associated RhoGEF (LARG), and then treated with Ang II, losartan, PD123319, or Val5-Ang II. The target mRNA and protein levels were determined using qPCR and Western blot analysis, respectively. Rat aortic rings were isolated, and the isometric contraction was measured with a force transducer and recorder.
Results: Stimulation with Ang II (0.1 μmol/L) for 0.5 h significantly increased the level of LARG mRNA in VSMCs. At 3, 6, and 9 h after the treatment with Ang II (0.1 μmol/L) plus AT2 antagonist PD123319 (1 μmol/L) or with AT1 agonist Val5-Ang II (1 μmol/L), the LARG protein, RhoA activity, and phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) in VSMCs were significantly increased. Knockdown of LARG with siRNA reduced these effects caused by AT1 receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished.
Conclusion: Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT1, thereby maintaining vascular tone.
Keywords:
Methods: Primary cultured VSMCs from Sprague-Dawley rats were transfected with siRNAs against leukemia-associated RhoGEF (LARG), and then treated with Ang II, losartan, PD123319, or Val5-Ang II. The target mRNA and protein levels were determined using qPCR and Western blot analysis, respectively. Rat aortic rings were isolated, and the isometric contraction was measured with a force transducer and recorder.
Results: Stimulation with Ang II (0.1 μmol/L) for 0.5 h significantly increased the level of LARG mRNA in VSMCs. At 3, 6, and 9 h after the treatment with Ang II (0.1 μmol/L) plus AT2 antagonist PD123319 (1 μmol/L) or with AT1 agonist Val5-Ang II (1 μmol/L), the LARG protein, RhoA activity, and phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) in VSMCs were significantly increased. Knockdown of LARG with siRNA reduced these effects caused by AT1 receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished.
Conclusion: Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT1, thereby maintaining vascular tone.