Abstract
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. 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 Val(5)-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. 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 AT(2) antagonist PD123319 (1 μmol/L) or with AT(1) agonist Val(5)-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 AT(1) receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished. Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT(1), thereby maintaining vascular tone.
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