Faculty Bibliography

OBJECTIVES/OBJECTIVE:This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism. METHODS:Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability. RESULTS:Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant. CONCLUSIONS:Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.

GPCR (G-protein-coupled receptor) signalling at the plasma membrane is under tight control. In the case of neuropeptides such as SP (substance P), plasma membrane signalling is regulated by cell-surface endopeptidases (e.g. neprilysin) that degrade extracellular neuropeptides, and receptor interaction with β-arrestins, which uncouple receptors from heterotrimeric G-proteins and mediate receptor endocytosis. By recruiting GPCRs, kinases and phosphatases to endocytosed GPCRs, β-arrestins assemble signalosomes that can mediate a second wave of signalling by internalized receptors. Endosomal peptidases, such as ECE-1 (endothelin-converting enzyme-1), can degrade SP in acidified endosomes, which destabilizes signalosomes and allows receptors, freed from β-arrestins, to recycle and resensitize. By disassembling signalosomes, ECE-1 terminates β-arrestin-mediated endosomal signalling. These mechanisms have been studied in model cell systems, and the relative importance of plasma membrane and endosomal signalling to complex pathophysiological processes, such as inflammation, pain and proliferation, is unclear. However, deletion or inhibition of metalloendopeptidases that control neuropeptide signalling at the plasma membrane and in endosomes has marked effects on inflammation. Neprilysin deletion exacerbates inflammation because of diminished degradation of pro-inflammatory SP. Conversely, inhibition of ECE-1 attenuates inflammation by preventing receptor recycling/resensitization, which is required for sustained pro-inflammatory signals from the plasma membrane. β-Arrestin deletion also affects inflammation because of the involvement of β-arrestins in pro-inflammatory signalling and migration of inflammatory cells. Knowledge of GPCR signalling in specific subcellular locations provides insights into pathophysiological processes, and can provide new opportunities for therapy. Selective targeting of β-arrestin-mediated endosomal signalling or of mechanisms of receptor recycling/resensitization may offer more effective and selective treatments than global targeting of cell-surface signalling.

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.

Chronic pancreatitis (CP) is a devastating disease characterized by persistent and uncontrolled abdominal pain. Our lack of understanding is partially due to the lack of experimental models that mimic the human disease and also to the lack of validated behavioral measures of visceral pain. The ligand-gated cation channel transient receptor potential ankyrin 1 (TRPA1) mediates inflammation and pain in early experimental pancreatitis. It is unknown if TRPA1 causes fibrosis and sustained pancreatic pain. We induced CP by injecting the chemical agent trinitrobenzene sulfonic acid (TNBS), which causes severe acute pancreatitis, into the pancreatic duct of C57BL/6 trpa1(+/+) and trpa1(-/-) mice. Chronic inflammatory changes and pain behaviors were assessed after 2-3 wk. TNBS injection caused marked pancreatic fibrosis with increased collagen-staining intensity, atrophy, fatty replacement, monocyte infiltration, and pancreatic stellate cell activation, and these changes were reflected by increased histological damage scores. TNBS-injected animals showed mechanical hypersensitivity during von Frey filament probing of the abdomen, decreased daily voluntary wheel-running activity, and increased immobility scores during open-field testing. Pancreatic TNBS also reduced the threshold to hindpaw withdrawal to von Frey filament probing, suggesting central sensitization. Inflammatory changes and pain indexes were significantly reduced in trpa1(-/-) mice. In conclusion, we have characterized in mice a model of CP that resembles the human condition, with marked histological changes and behavioral measures of pain. We have demonstrated, using novel and objective pain measurements, that TRPA1 mediates inflammation and visceral hypersensitivity in CP and could be a therapeutic target for the treatment of sustained inflammatory abdominal pain.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with beta-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and beta-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/beta-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from beta-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained beta-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.