Senthilkumar Rajagopal

Release of GLP-1 and PYY in response to the activation of G protein-coupled bile acid receptor TGR5 is mediated by Epac/PLC-ε pathway and modulated by endogenous H2S

Activation of plasma membrane TGR5 receptors in enteroendocrine cells by bile acids is known to regulate gastrointestinal secretion and motility and glucose homeostasis. The endocrine functions of the gut are modulated by microenvironment of the distal gut predominantly by sulfur-reducing bacteria of the microbiota that produce H2S. However, the mechanisms involved in the release of peptide hormones, GLP-1 and PYY in response to TGR5 activation by bile acids and the effect of H2S on bile acid-induced release of GLP-1 and PYY are unclear. In the present study, we have identified the signaling pathways activated by the bile acid receptor TGR5 to mediate GLP-1 and PYY release and the mechanism of inhibition of their release by H2S in enteroendocrine cells. The TGR5 ligand oleanolic acid (OA) stimulated Gαs and cAMP formation, and caused GLP-1 and PYY release. OA-induced cAMP formation and peptide release were blocked by TGR5 siRNA. OA also caused an increase in PI hydrolysis and intracellular Ca2+. Increase in PI hydrolysis was abolished in cells transfected with PLC-ε siRNA. 8-pCPT-2′-O-Me-cAMP, a selective activator of Epac, stimulated PI hydrolysis, and GLP-1 and PYY release. L-Cysteine, which activates endogenous H2S producing enzymes cystathionine-γ-lyase and cystathionine-β-synthase, and NaHS and GYY4137, which generate H2S, inhibited PI hydrolysis and GLP-1 and PYY release in response to OA or 8-pCPT-2′-O-Me-cAMP. Propargylglycine, an inhibitor of CSE, reversed the effect of L-cysteine on PI hydrolysis and GLP-1 and PYY release. We conclude: (i) activation of Gαs-coupled TGR5 receptors causes stimulation of PI hydrolysis, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε/Ca2+ pathway, and (ii) H2S has potent inhibitory effects on GLP-1 and PYY release in response to TGR5 activation, and the mechanism involves inhibition of PLC-ε/Ca2+ pathway.

Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

Cytokine-Induced S-Nitrosylation of Soluble Guanylyl Cyclase and Expression of Phosphodiesterase 1A Contribute to Dysfunction of Longitudinal Smooth Muscle Relaxation

The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP–phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)–induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.

Inhibition of RhoA-dependent pathway and contraction by endogenous hydrogen sulfide in rabbit gastric smooth muscle cells

Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase (MLCP). H2S produced as an endogenous signaling molecule synthesized mainly from l-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. The aim of this study was to analyze the expression of CSE and H2S function in the regulation of MLCP activity, 20-kDa regulatory light chain of myosin II (MLC20) phosphorylation, and contraction in isolated gastric smooth muscle cells. Both mRNA expression and protein expression of CSE, but not CBS, were detected in smooth muscle cells of rabbit, human, and mouse stomach. l-cysteine, an activator of CSE, and NaHS, a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase-sensitive phosphorylation of MYPT1, PKC-sensitive phosphorylation of CPI-17, and MLC20 phosphorylation and sustained muscle contraction. The inhibitory effects of l-cysteine, but not NaHS, were blocked upon suppression of CSE expression by siRNA or inhibition of its activity by dl-propargylglycine (PPG) suggesting that the effect of l-cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels, had no effect on the inhibition of contraction by H2S. Both l-cysteine and NaHS had no effect on basal cAMP and cGMP levels but augmented forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both endogenous and exogenous H2S inhibit muscle contraction, and the mechanism involves inhibition of Rho kinase and PKC activities and stimulation of MLCP activity leading to MLC20 dephosphorylation and inhibition of muscle contraction.

202 Hydrogen Sulfide (H2S) Inhibits Bile Acid Receptor TGR5-Mediated GLP-1 Release From Enteroendrocrine Cells: Possible Involvement of H2S in Altered Glucose Metabolism in Diabetes

G A A b st ra ct s gene expression was lower in the Ift88ΔS mice. Despite the rapid induction of gastrin secretion, Ift88ΔS mice showed delayed induction of acid secretion with food intake, and lower mRNA levels of both H+K+ATPase α and β-subunits. In these mice, gastrin, ghrelin, and somatostatin gene expression did not change in response to food intake as in controls. At age 5 months, Ift88ΔS stomachs were histologically normal. However when analyzed at 8 months, mice developed gastric pathology associated with endocrine dysfunction including foveolar hyperplasia, hypergastrinemia, and hypochlorhydria. Epithelial deletion of Kif3a, another gene required for cilia formation, recapitulated the phenotypes observed in Ift88ΔS mice. Conclusion: Overall, our results demonstrated that gastric epithelial cilia regulate endocrine cell function and gastric homeostasis by sensing luminal food.