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

Senthilkumar Rajagopal, Divya P Kumar, Sunila Mahavadi, Sayak Bhattacharya, Ruizhe Zhou, Carlos U Corvera, Nigel William Bunnett, John R Grider, Karnam S Murthy

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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 Gas, but not Gaq Gai1, Gai2, or Gai3, 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 Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.
Original languageEnglish
Pages (from-to)G527 - G535
Number of pages9
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume304
Issue number5
DOIs
Publication statusPublished - 2013

Cite this

Rajagopal, Senthilkumar ; Kumar, Divya P ; Mahavadi, Sunila ; Bhattacharya, Sayak ; Zhou, Ruizhe ; Corvera, Carlos U ; Bunnett, Nigel William ; Grider, John R ; Murthy, Karnam S. / 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. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 2013 ; Vol. 304, No. 5. pp. G527 - G535.
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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 Gas, but not Gaq Gai1, Gai2, or Gai3, 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 Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.",
author = "Senthilkumar Rajagopal and Kumar, {Divya P} and Sunila Mahavadi and Sayak Bhattacharya and Ruizhe Zhou and Corvera, {Carlos U} and Bunnett, {Nigel William} and Grider, {John R} and Murthy, {Karnam S}",
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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. / Rajagopal, Senthilkumar; Kumar, Divya P; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U; Bunnett, Nigel William; Grider, John R; Murthy, Karnam S.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 304, No. 5, 2013, p. G527 - G535.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - 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

AU - Rajagopal, Senthilkumar

AU - Kumar, Divya P

AU - Mahavadi, Sunila

AU - Bhattacharya, Sayak

AU - Zhou, Ruizhe

AU - Corvera, Carlos U

AU - Bunnett, Nigel William

AU - Grider, John R

AU - Murthy, Karnam S

PY - 2013

Y1 - 2013

N2 - 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 Gas, but not Gaq Gai1, Gai2, or Gai3, 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 Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

AB - 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 Gas, but not Gaq Gai1, Gai2, or Gai3, 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 Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

UR - http://ajpgi.physiology.org/content/304/5/G527

U2 - 10.1152/ajpgi.00388.2012

DO - 10.1152/ajpgi.00388.2012

M3 - Article

VL - 304

SP - G527 - G535

JO - American Journal of Physiology - Gastrointestinal and Liver Physiology

JF - American Journal of Physiology - Gastrointestinal and Liver Physiology

SN - 0193-1857

IS - 5

ER -