We report the unexpected localization of GpBAR1-IR in myenteric and submucosal neurons of the mouse intestine. A large proportion of GpBAR1-expressing neurons of the myenteric plexus coexpressed NOS and are thus inhibitory motor neurons or descending interneurons, and the majority of these neurons expressed GpBAR1-IR. Consistent with this localization, a BA that is a potent GpBAR1 agonist inhibited motility of intestinal segments by a neurogenic, nitrergic mechanism, and delayed gastric emptying and intestinal transit. Our results reveal a novel function for BAs and GpBAR1 in the enteric nervous system, and provide a mechanistic explanation for the well-known actions of BAs on intestinal motility.
We detected GpBAR1-IR and mRNA throughout the gastrointestinal tract, with distinct regional differences. GpBAR1 mRNA was highly expressed in the mouse gall bladder, liver and small and large intestine, including separated muscularis externa
-myenteric plexus and submucosa-submucosal plexus. Lower levels were detected in the stomach and GpBAR1 was undetectable in the esophagus. These results agree with GpBAR1 mRNA expression in human tissues (13
). We observed regional differences in GpBAR1-IR in the small and large intestine, with prominent localization in the mucosa and muscularis externa of the small intestine but not the colon. Although GpBAR1-IR was present in enterocytes of the small intestine, it was also present in unidentified cells in the lamina propria that may be macrophages or monocytes (12
). We did not detect GpBAR1-IR in the colonic mucosa, which agrees with its lack of expression in colonic epithelial cell lines (13
). However, GpBAR1 mRNA is present in cell lines derived from intestinal endocrine cells, where activation stimulates hormone release (13
). It will be important to determine whether GpBAR1 is present in endocrine cells of the small and large intestine, where activation could control the secretion of hormones that regulate motility and gall bladder contraction.
The major unexpected finding of our study is the prominent localization of GpBAR1 in enteric neurons. GpBAR1-IR was present in approximately half of all myenteric neurons of the intestine. Within the myenteric plexus, most NOS-positive inhibitory neurons (>80%) expressed GpBAR1-IR, and a high proportion of GpBAR1-positive neurons coexpressed NOS, especially in the large intestine (49–60%). Detection of GpBAR1-IR in NOS-positive inhibitory neurons of the myenteric plexus suggests that these neurons mediate the inhibitory actions of BAs on intestinal motility. Supporting this suggestion, DCA, a potent agonist of GpBAR1, strongly inhibited spontaneous contractions of longitudinal muscle from the proximal colon, whereas UDCA, a weak agonist, had little effect. Although DCA inhibited contractions of the proximal colon and the ileum, the effect was far more marked in the colon. This difference is unlikely to reflect the minor differences in the proportion of GpBAR1-IR neurons coexpressing inhibitory or excitatory transmitters between the proximal colon and the ileum. A more likely explanation is the presence of GpBAR1-IR in muscle cells of the ileum but not the colon, allowing BAs to directly affect ileal muscle. In both tissues, tetrodotoxin and L-NAME abolished the effects of DCA. BAs also inhibit contractions of isolated rabbit and guinea pig ileum (29
). Our results are consistent with the novel hypothesis that BAs activate GpBAR1 on inhibitory motor neurons to release NO, which inhibits spontaneous contractions of the intestine. This may represent a physiologically important mechanism, since luminal administration of DCA also inhibited gastric emptying and delayed intestinal transit in intact mice. The source of NO produced cannot be directly determined using our current methodology, and it may originate from non-neuronal cells (34
). Although bile acids can activate muscarinic receptors (35
), our data demonstrating that DCA-evoked inhibitory effects on contractile activity were unaffected by atropine indicates that muscarinic receptors are not involved in these reponses. BAs may also act on the mucosa to release factors that control motility. The possibility that bile acids mediate their effects through a GpBAR1-independent mechanism can only be directly examined once GpBAR1-specific antagonists and GpBAR1-deficient mice become readily available.
Our findings may explain the well-known effects of luminal BAs on intestinal motility. BA infusion into the human jejunum or ileum inhibits transit, suggesting the existence of intestinal BA receptors (26
). Thus, BAs may contribute to the “ileal brake”, by which BAs and lipids slow transit to permit complete digestion and absorption (27
). Our observations that GpBAR1-IR is expressed by inhibitory motor neurons, and that a GpBAR1 agonist inhibits contractions by a neurogenic mechanism and delays transit, suggest that GpBAR1 mediates the ileal brake. Studies of GpBAR1-deficient mice are required to further evaluate this possibility. The mechanism by which luminal BAs activate neuronal GpBAR1 also remains to be determined. Luminal BAs could be transported across the epithelium through specific bile acid transporters to activate GpBAR1 on enteric nerve fibers or smooth muscle cells (37
), through a similar mechanism to that through which glucose affects myenteric neurons (38
). The more hydrophobic bile acids, DCA and CDCA can more readily traverse the apical and basolateral plasma membrane bilayers (39
), thereby, theoretically crossing the epithelium to reach submucosal or myenteric neurons. The inability of TDCA to inhibit contractile activity of the isolated ileum suggests that this hydrophilic bile acid does not effectively access target neurons within the small intestine. Luminal BAs may also affect the release of hormones from entero-endocrine cells, since hormonal mechanisms may also account for the BA-induced ileal brake (29
). Alternatively, BAs from the circulation may activate the receptor on enteric neurons or other cell types. GpBAR1-IR was colocalized with NFM-IR, which has been characterized as a marker of IPANs in the mouse small intestine (33
). IPANs innervate the mucosa and other neurons within the enteric circuitry (40
), thus it is possible that BAs mediate their effects through specific activation of these neurons.
We detected GpBAR1-IR in intestinal submucosal neurons, many of which contain ChAT and are cholinergic secretomotor neurons or intrinsic primary afferent neurons (33
). It remains to be determined if non-cholinergic secretomotor neurons containing vasoactive intestinal peptide also express GpBAR1. BAs stimulate secretion of fluid and mucus by a neuronal, cholinergic mechanism (20
), which may also depend on NO generation (41
). Our localization of GpBAR1-IR to cholinergic submucosal neurons raises the possibility that GpBAR1 mediates these effects of BAs on intestinal secretion. Thus, GpBAR1-expressing secretomotor neurons might function as chemo-sensors, stimulating secretory reflexes as a defensive mechanism to eliminate BAs and other noxious agents. However, BAs may directly activate GpBAR1 or other bile acid receptors on enterocytes to regulate fluid and electrolyte secretion. Activation of GpBAR1 in the gall bladder epithelium stimulates the cystic fibrosis transmembrane conductor regulator chloride channel to promote chloride secretion (14
). Additional studies are required to define the role of GpBAR1 in BA-induced secretion in the intestine.
In conclusion, we have described the unique localization of the BA receptor GpBAR1 in the enteric nervous system, where the receptor is suitably located to mediate the effects of BAs on intestinal motility and secretion. This report adds to the emerging role of GpBAR1 in control of energy expenditure, weight gain, blood flow, bile acid homeostasis and immune responses.