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.

TGR5, Not Only a Metabolic Regulator

“…TGR5 activates AKT (Kida et al, 2013; Perino et al, 2014), TRPA1 (Lieu et al, 2014), and Epac (Kumar et al, 2012) pathways. And it inhibits NF-κB (Pols et al, 2011; Wang et al, 2011; Yoneno et al, 2013), STAT3 (Guo et al, 2015b), and RhoA/Rho kinase (Rajagopal et al, 2013) pathways. TGR5 has opposite functions in ERK1/2 pathway.…”

The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis: Molecular Insights for Targeting Disorders of Sensation and Inflammation

“…Bile Acids. Bile acids are essential for the digestion and absorption of dietary fat and can also activate the bile acid receptor TGR5 (GPBA), a GPCR that couples to Ga s , adenylyl cyclase, and cAMP production (Kawamata et al, 2003;Rajagopal et al, 2013). Bile acids are markedly increased in the circulation and tissues of patients with cholestatic liver disease and may contribute to the chronic and intractable pruritus that is a common feature of this condition (Hayashi and Majima, 1999).…”

Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/β-arrestin activation of ERK1/2 and Src kinase

“…Total RNA was extracted from cultured gastric smooth muscle cells by treatment with RNaqueous reagent and contaminant genomic DNA removed by treatment with TURBO DNase as described previously (23). RNA (5 g) was reversibly transcribed and amplified by PCR under standard conditions.…”

“…Wild-type GRK2, GRK5, RGS4, kinase-deficient GRK2(K220R) and GRK5(K215R), phosphorylation-deficient RGS4(S103A/S108A), and ␤-arrestin1 and -2 siRNA were subcloned into the multiple cloning site (EcoRI) of the eukaryotic expression vector pcDNA3. Recombinant plasmid cDNAs were transiently transfected into smooth muscle cultures for 48 h. The cells were cotransfected with 2 g pcDNA3 vector and 1 g of pGreen Lantern-1 DNA to monitor transfection efficiency (23).…”