title: Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway.
abstract: The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0551, USA.
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Rajagopal
authors.forename: Divya P
authors.initials: DP
authors.lastname: Kumar
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Sayak
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Ruizhe
authors.initials: R
authors.lastname: Zhou
authors.forename: Carlos U
authors.initials: CU
authors.lastname: Corvera
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
authors.forename: John R
authors.initials: JR
authors.lastname: Grider
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009126:Muscle Relaxation / Q000502:physiology; D009130:Muscle, Smooth / Q000502:physiology*; D009828:Oleanolic Acid / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D054460:rho-Associated Kinases / Q000378:metabolism / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*
chemical_list: C482601:Epac protein, mouse; C513561:Gpbar1 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D009828:Oleanolic Acid; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D020742:rhoA GTP-Binding Protein