Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

Abstract

Background: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract.

Methods: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility.

Key results: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit.

Conclusions & inferences: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

Figure 1. GpBAR1 antibody characterization

A. BA-stimulated cAMP generation. HEK-GpBAR1-GFP and HEK-VC cells were incubated with vehicle (0.1% ethanol, EtOH), forskolin (10 nM) or BAs (100 µM) for 5 min. cAMP generation is expressed in pmol per well (n=6). B. Localization of GpBAR1-IR by immunofluorescence. In HEK-GpBAR1-GFP cells, GFP and GpBAR1-IR colocalized at the plasma membrane and in vesicles (upper row). Preadsorption (+ peptide) abolished signal for GpBAR1-IR but not GFP (middle row). Neither GFP nor GpBAR1-IR was detected in HEK-VC cells (bottom row). Scale bar = 10 µm. C. Detection of GpBAR1-IR by Western blotting. Membranes from HEK-GpBAR1-GFP or HEK-VC cells were separated by SDS-PAGE. The same blot was probed with antibodies to GFP (left) or GpBAR1 (middle) (right lanes) or preadsorbed antibody to GpBAR1 (left lanes, + peptide). Merged images are in right panel. In membranes from HEK-GpBAR1-GFP cells, antibodies to GFP and GpBAR1 detected proteins of ~60–70 KDa, corresponding to the GpBAR1-GFP fusion protein (merged). In membranes from HEK-VC cells, GFP-IR was undetectable and GpBAR1-IR was markedly diminished. Preadsorpion of GpBAR1 antibody did not affect GFP-IR but suppressed GpBAR1-IR.

Figure 2. Localization of GpBAR1-IR in sections of mouse stomach and small and large intestine

a: Representative examples of GpBAR1-IR in the wall of the gastric corpus (A, B, C) and antrum (D). GpBAR1-IR was expressed in the myenteric plexus (MP). Circular muscle, CM; longitudinal muscle, LM. Preadsorption of the GpBAR1 antibody with immunizing peptide abolished GpBAR1-IR (C). b: Representative examples of GpBAR1-IR in the wall of the duodenum (A), ileum (B), cecum (C) and colon (D) are shown. GpBAR1-IR was expressed in the myenteric plexus (MP) and submucosal plexus (SMP) of the small and large intestine. GpBAR1-IR was more highly expressed in the mucosa, muscularis externa (circular muscle, CM; longitudinal muscle, LM), of the small intestine compared to the large intestine. Preadsorption of the GpBAR1 antibody with immunizing peptide abolished GpBAR1-IR.

Figure 3. Amplification of GpBAR1 mRNA in gastrointestinal tissues

A 449 bp product corresponding to GpBAR1 was amplified and identified by sequence analysis. A. GpBAR1 was amplified from gall bladder, liver and cecum, with low levels in stomach and no detectable product in esophagus (Esoph.). B, C. GpBAR1 was highly expressed in dissected muscularis externa-myenteric plexus (ME-MP) and expressed at lower levels in dissected submucosa-submucosal plexus (SM-SMP) of duodenum (Duod.), ileum, proximal colon (Prox. Co.) and distal colon (Dist. Co.). No products were detected without reverse transcriptase (RT).

Figure 4. Localization of GpBAR1-IR and PGP9.5-IR in whole mounts of myenteric plexus

GpBAR1-IR (left panels) was detected in approximately half of all myenteric neurons expressing PGP9.5-IR (middle panels) in the ileum (A), cecum (B) and distal colon (C). Merged images (right panels) demonstrate colocalization of GpBAR1-IR and PGP9.5-IR (arrowheads). Although cells expressing GpBAR1-IR almost always expressed PGP9.5-IR, some PGP9.5-IR positive neurons did not express GpBAR1-IR (arrowheads with asterisk). D. Preadsorption of the GpBAR1 antibody with immunizing peptide abolished GpBAR1-IR in the ileum. Scale bars = 50 µm.

Figure 5. Localization of GpBAR1-IR and NOS-IR in whole mounts of myenteric plexus

Most (>80%) neurons expressing GpBAR1-IR (left panels) also expressed NOS-IR (middle panels) in the duodenum (A), ileum (B), cecum (C) and proximal colon (D). Merged images (right panels) demonstrate colocalization of GpBAR1-IR and NOS-IR (arrowhead). Neurons expressing only GpBAR1-IR or NOS-IR are indicated (arrowheads with asterisk). Scale bars = 50 µm.

Figure 6. Localization of GpBAR1-IR, PGP9.5-IR and ChAT-IR in whole mounts of submucosal plexus

Localization of GpBAR1-IR (left panels) and PGP9.5-IR (middle panels) (A) or ChAT-IR (middle panels) (B) in duodenum and distal colon. Merged images (right panels) indicate invariable colocalization of GpBAR1-IR and PGP9.5-IR, and frequent colocalization of GpBAR1-IR and ChAT-IR (arrowheads). Some neurons expressing GpBAR1-IR did not express ChAT-IR (arrowheads with asterisk). Scale bars = 20 µm.

Figure 7. Effects of BAs on spontaneous contractile activity of longitudinal muscle of the proximal colon and ileum

A. Representative recordings from proximal colon. DCA (100 µM) immediately inhibited spontaneous phasic contractions, but did not alter basal tone. UDCA (100 µM) did not reduce the frequency of spontaneous contractions. Tetrodotoxin (TTX) and L-NAME abolished the effects of DCA. B–D. Effects of treatments on frequency of contraction, in which the frequency prior to stimulation is normalized to 1. B. Effects of graded concentrations on DCA in proximal colon. C, D. Tetrodotoxin and L-NAME abolished the effects of DCA in the proximal colon (C) and ileum (D). The effects of DCA (100 µM) on the proximal colon were significantly reduced by hexamethonium, but not by atropine (C). TDCA (100 µM) did not have any effect on spontaneous activity of the ileum relative to vehicle control (D). * P< 0.05, ** P<0.01, *** P<0.001 (n≥6).

Figure 8. Effects of BAs on gastric emptying and small intestinal transit

Phenol red and DCA, UCDA or vehicle (control) were administered by gavage. After 30 min, the proportion of the administered phenol red was determined in the stomach and in 3 equal segments of small intestine. Gastric emptying (A), transit of the marker in the intestinal segments (B) and the geometric center of the marker in the small intestine (C) were determined. Compared to control, DCA but not UCDA inhibited gastric emptying and caused retention of the marker in the proximal segments of the intestine, reducing the geometric mean of the marker distribution. * P< 0.05 (n≥6).