The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice

Abstract

Background & aims: Abnormal delivery of bile acids (BAs) to the colon as a result of disease or therapy causes constipation or diarrhea by unknown mechanisms. The G protein-coupled BA receptor TGR5 (or GPBAR1) is expressed by enteric neurons and endocrine cells, which regulate motility and secretion.

Methods: We analyzed gastrointestinal and colon transit, as well as defecation frequency and water content, in wild-type, knockout, and transgenic mice (trg5-wt, tgr5-ko, and tgr5-tg, respectively). We analyzed colon tissues for contractility, peristalsis, and transmitter release.

Results: Deoxycholic acid inhibited contractility of colonic longitudinal muscle from tgr5-wt but not tgr5-ko mice. Application of deoxycholic acid, lithocholic acid, or oleanolic acid (a selective agonist of TGR5) to the mucosa of tgr5-wt mice caused oral contraction and caudal relaxation, indicating peristalsis. BAs stimulated release of the peristaltic transmitters 5-hydroxytryptamine and calcitonin gene-related peptide; antagonists of these transmitters suppressed BA-induced peristalsis, consistent with localization of TGR5 to enterochromaffin cells and intrinsic primary afferent neurons. tgr5-ko mice did not undergo peristalsis or transmitter release in response to BAs. Mechanically induced peristalsis and transmitter release were not affected by deletion of tgr5. Whole-gut transit was 1.4-fold slower in tgr5-ko than tgr5-wt or tgr5-tg mice, whereas colonic transit was 2.2-fold faster in tgr5-tg mice. Defecation frequency was reduced 2.6-fold in tgr5-ko and increased 1.4-fold in tgr5-tg mice compared with tgr5-wt mice. Water content in stool was lower (37%) in tgr5-ko than tgr5-tg (58%) or tgr5-wt mice (62%).

Conclusions: The receptor TGR5 mediates the effects of BAs on colonic motility, and deficiency of TGR5 causes constipation in mice. These findings might mediate the long-known laxative properties of BAs, and TGR5 might be a therapeutic target for digestive diseases.

Figure 1.. TGR5-dependent regulation of colonic contractility.

Recordings were made of spontaneous phasic contractions of longitudinal muscle of isolated proximal colon. A. Representative recordings from tgr5-wt and tgr5-ko mice. In tgr5-wt mice, UDCA (100 μΜ) had no effect, whereas DCA (100 μΜ) immediately inhibited spontaneous phasic contractions. In tgr5-ko mice, DCA was inactive. B, C. Mean results of the effects of DCA on frequency (B) and tension (C) normalized to basal values (1.0). DCA reduced the frequency and tension in tgr5-wt but not in tgr5-ko mice. * P< 0.05, *** P<0.001 to tgr5-wt.

Figure 2.. TGR5-dependent stimulation of colonic peristalsis.

Peristaltic contractions of isolated proximal colon were recorded from tgr5-wt and tgr5-ko mice. A. Representative recordings of ascending contraction and descending relaxation to mucosal application of DCA (100 μΜ), which stimulated peristalsis in tgr5-wt mice, and had diminished effects in tgr5-ko mice. B, C. Mean results of ascending contraction and descending relaxation (grams force above or below baseline tone) in response to mucosal application of graded concentrations of DCA (1– 100 μΜ) (B) or graded mechanical stimulation of the mucosa (2–8 strokes). Compared to responses in tgr5-wt mice, peristaltic contractions to DCA were abolished or attenuated in tgr5-ko mice, whereas responses to mechanical stimulation were unaffected by TGR5 expression. **P<0.005 to tgr5-wt.

Figure 3.. Contributions of 5-HT and CGRP to colonic peristalsis.

Peristaltic contractions of isolated proximal colon were recorded from C57BL/6 mice. A. Representative recordings of ascending contraction and descending relaxation showing that GR113808 or CGRP8–37 attenuated DCA (100 pM)-stimulated peristalsis. B, C. Mean results of ascending contraction and descending relaxation to graded concentrations of DCA (B) or LCA (C) (1–100 μΜ). GR113808 or CGRP8–37 attenuated DCA- and LCA-stimulated peristalsis, although CGRP8–37 was more effective. *P<0.05, **P<0.005, ***P<0.001 to vehicle.

Figure 4.. TGR5-dependent release of peristaltic transmitters from proximal colon.

Release of 5-HT-IR (A) or CGRP-IR (B) into the central compartment of the isolated proximal colon from tgr5-wt and trg5-ko mice after mucosal application of DCA (1–100 μΜ) or OA (100 μΜ). Whereas DCA and OA stimulated 5-HT and CGRP release in tgr5-wt mice, responses were absent or attenuated in tgr5-ko mice. *P<0.05, **P<0.01 to basal.

Figure 5.. Localization of TGR5-IR to myenteric neurons and EC cells of mouse colon.

TGR5 was detected in C57BL/6 mice using antibody NLS1937 (A) or P87/8 8 (B, C) with similar results. A. In sections of whole thickness colon, TGR5-IR was detected in neurons of the myenteric plexus (MP) within the muscularis externa (ME) and in mucosal epithelial cells (arrowheads). CGRP-IR colocalized with TGR5-IR in myenteric neurons, and was also found in nerve fibers in the mucosa (arrowhead asterisk). B. In sections of the colonic mucosa, TGR5-LI colocalized with 5-HT-IR in EC cells (arrowheads), and was also detected in epithelial cells. C. Analysis of the neurochemical coding of neurons in wholemounts of the myenteric plexus revealed colocalization of TGR5-IR, CGRP-IR and NFM-IR in IPANS (arrowheads), although TGR5-IR was also detected in other neuronal subtypes (arrowhead asterisk). Scale bars: A 100 μm, B, C 20 μm.

Figure 6.. Gastrointestinal and colonic transit.

A. Whole gut transit of Evans blue dye. After gavage of dye, the time for expulsion of the first blue pellet was longer in tgr5-ko mice than tgr5-wt or tgr5-tg mice, indicating delayed transit. B. Expulsion of a glass bead from the colon. After insertion of a glass bead into the colon, the time for expulsion was less in tgr5-tg mice than in tgr5-wt or tgr5-ko mice, indicating accelerated colonic transit. **P< 0.01, ***P<0.001 to tgr5-wt.

Figure 7.. Defecation frequency and water content.

A. Defecation frequency was diminished in tgr5-ko mice but increased in trg5-tg mice compared to tgr5-wt mice. B. Fecal water content was diminished in tgr5-ko mice compared to tgr5-wt or tgr5-tg mice. **P< 0.01, ***P<0.001 to tgr5-wt.