Rho activation in excitatory agonist-stimulated vascular smooth muscle

Small GTPase Rho and its downstream effector, Rho kinase, have been implica ted in agonist-stimulated Ca2+ sensitization of 20-kDa myosin light chain ( MLC20) phosphorylation and contraction in smooth muscle. In the present stu dy we demonstrated for the first time that excitatory receptor agonists ind uce 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 Rho A-binding protein, Rhotekin, we found that a thromboxane A(2) mimetic, U-46 619, which induced a sustained contractile response, induced a sustained ri se 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 activati on was thromboxane A(2) receptor-mediated and reversible. Other agonists in cluding 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 kin ase inhibitor genistein substantially inhibited RhoA activation by these ag onists, except for ET-1. Thus excitatory agonists induce Rho activation in an agonist-specific manner, which is thought to contribute to stimulation o f MLC20 phosphorylation Ca2+ sensitivity.