Rho activation in excitatory agonist-stimulated vascular smooth muscle

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Abstract
Small GTPase Rho and its downstream effector, Rho kinase, have been implicated in agonist-stimulated Ca2+ sensitization of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. In the present study we demonstrated for the first time that excitatory receptor agonists induce increases in amounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aortic smooth muscle. Using a pull-down assay with a recombinant RhoA-binding protein, Rhotekin, we found that a thromboxane A2 mimetic, U-46619, which induced a sustained contractile response, induced a sustained rise in the amount of GTP-RhoA in a dose-dependent manner with an EC50 value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxane A2 receptor-mediated and reversible. Other agonists including norepinephrine, serotonin, histamine, and endothelin-1 (ET-1) also stimulated RhoA, albeit to lesser extents than U-46619. In contrast, ANG II and phorbol 12,13-dibutyrate failed to increase GTP-RhoA. The tyrosine kinase inhibitor genistein substantially inhibited RhoA activation by these agonists, except for ET-1. Thus excitatory agonists induce Rho activation in an agonist-specific manner, which is thought to contribute to stimulation of MLC20 phosphorylation Ca2+ sensitivity.

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