Reduction of epithelial secretion in male rat distal colonic mucosa by bile acid receptor TGR5 agonist, INT-777: role of submucosal neurons - PubMed (original) (raw)

. 2016 Nov;28(11):1663-1676.

doi: 10.1111/nmo.12866. Epub 2016 Jun 3.

Affiliations

Reduction of epithelial secretion in male rat distal colonic mucosa by bile acid receptor TGR5 agonist, INT-777: role of submucosal neurons

Henri Duboc et al. Neurogastroenterol Motil. 2016 Nov.

Abstract

Background: Recent evidence from rat neuron-free mucosa study suggests that the membrane bile acid receptor TGR5 decreases colonic secretion under basal and stimulated conditions. As submucosal neurons are key players in secretory processes and highly express TGR5, we investigated their role in TGR5 agonist-induced inhibition of secretion and the pathways recruited.

Methods: TGR5 expression and localization were assessed in rat proximal (pC) and distal (dC) colon by qPCR and immunohistochemistry with double labeling for cholinergic neurons in whole-mount preparations. The influence of a selective (INT-777) or weak (ursodeoxycholic acid, UDCA) TGR5 agonist on colonic secretion was assessed in Ussing chambers, in dC preparation removing seromuscular ± submucosal tissues, in the presence of different inhibitors of secretion pathways.

Key results: TGR5 mRNA is expressed in full thickness dC and pC and immunoreactivity is located in colonocytes and pChAT-positive neurons. Addition of INT-777, and less potently UDCA, decreased colonic secretion in seromuscular stripped dC by -58.17± 2.6%. INT-777 effect on basal secretion was reduced in neuron-free and TTX-treated mucosal-submucosal preparations. Atropine, hexamethonium, indomethacin, and L-NAME all reduced significantly INT-777's inhibitory effect while the 5-HT4 antagonist, RS-39604, and lidocaine abolished it. INT-777 inhibited stimulated colonic secretion induced by nicotine, but not cisapride, carbachol or PGE2.

Conclusions & inferences: TGR5 activation inhibits basal and stimulated distal colonic secretion in rats by acting directly on epithelial cells and also inhibiting submucosal neurons. This could represent a counter-regulatory mechanism, at the submucosal level, of the known prosecretory effect of bile acids in the colon.

Keywords: TGR5; Ussing chambers; bile acids; distal colon; secretion; submucosal neurons.

© 2016 John Wiley & Sons Ltd.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Expression and distribution of TGR5 mRNA and protein in rat colonic tissue. (A) Comparison of TGR5 mRNA expression in distal and proximal rat colon by semi-quantitative qPCR (right). Data are means ± SEM, n as indicated in columns. **p<0.01 vs proximal colon, unpaired t test. (B) Localization of TGR5 protein expression in distal and proximal colon tissue sections by immunofluorescence. TGR5 immunofluorescence is located in cells of the epithelia, with staining visible both on the apical and basolateral sides of epithelial cells mostly in crypts and in submucosal and myenteric neurons. Scale: 100 μM. (C) Co-localization (yellow) of TGR5 (green) and pChAT (red) positive neurons in submucosal plexus of proximal (upper panel) and distal (lower panel) colon. Scale: 100 μM.

Figure 2

Figure 2

Dose-dependent effect of the selective TGR5 agonist, INT-777, on short-circuit current (Isc, in % baseline) (A) and transepithelial electrical resistance (TEER, in % baseline) (B) in seromuscular stripped preparations of rat distal colon mounted in Ussing chambers. INT-777 administered on the serosal side significantly reduced the Isc starting at the dose of 10 μM, and increased the TEER at the dose of 60 and 100 μM. Data are means ± SEM, n as indicated in parenthesis. *p<0.05 vs DMSO group, two-way ANOVA followed by Bonferroni's multiple comparison post hoc test.

Figure 3

Figure 3

Influence of a low affinity endogenous agonist for TGR5 receptor, ursodeoxycholic acid (UDCA) on short-circuit current (Isc, in % baseline) (A) and transepithelial electrical resistance (TEER, in % baseline) (B) in seromuscularly-stripped preparations of rat distal colon mounted in Ussing chambers. UDCA administered on the serosal side at 60 μM reduced significantly the Isc at 35 min (A), but had no effect on TEER (4D). Data are means ± SEM, n as indicated in parenthesis. *p<0.05 (60 μM) and #p<0.05 (100 μM) vs DMSO group, two-way ANOVA followed by Bonferroni's multiple comparison post hoc test.

Figure 4

Figure 4

Contribution of the submucosal plexus and epithelial cells in the selective TGR5 agonist (INT-777) induced inhibition of basal Isc. (A) In seromuscular stripped preparations of rat distal colon mounted in Ussing chambers INT-777-induced Isc inhibition is partially reduced by TTX pretreatment (1 μM) and abolished by pretreatment with the sodium channel blocker, lidocaine (1 μM). The inhibitory effect of INT-777 in neuron-free preparations of distal colon is significantly less than in preparations containing enteric nerves. DMSO does not affect the Isc in distal colon preparations with or without enteric neurons. Representative graphs of pooled Ussing experiments: data are means ± SEM, n as indicated in columns. *p<0.05, ***p<0.001 vs INT-777 treated group, unpaired t test. (B) Representative graphs from Isc recordings in Ussing chambers, showing the influence of INT-777 (100 μM) in neuron-containing distal colon preparations pretreated with TTX or lidocaine, and in neuron-free rat distal colonic tissue.

Figure 5

Figure 5

TGR5 agonist (INT-777) inhibition of basal secretion induced by several endogenously-released segretagogues in rat distal colon. Influence of INT-777 on Isc in seromuscular peeled preparations pretreated with the indomethacin (10 μM), atropine (10 μM), hexamethonium (100 μM), 5-HT4 antagonist (RS-39604, 10 μM), L-NAME (100 μM), or DMSO (10 μl). Data are means ± SEM, n as indicated in columns. *p<0.05, **p<0.01, ***p<0.001 vs INT-777 treated group, unpaired t test.

Figure 6

Figure 6

Influence of TGR5 activation on stimulated secretion: a role for nicotinic cholinergic submucosal neurons. Representative graphs of INT-777 inhibition of stimulated secretion. (A) INT-777 (100 μM) pretreatment reduced basal Isc, but did not affect the increase in secretion mediated by the 5-HT4 agonist, cisapride (10 μM). (B) In the presence of indomethacin (10 μM), INT-777 (100 μM) still reduced the basal Isc, but did not prevent PGE2 (10 μM)-induced secretion. In contrast, nicotine (100 μM)-induced secretion was reduced by INT-777 pretreatment. (C) Inhibition of nicotine-induced secretion by INT-777 was reproduced in a fresh preparation. Data are means ± SEM, n as indicated in columns. **p<0.01 vs nicotine-treated group, paired t test.

References

    1. Bajor A, Gillberg PG, Abrahamsson H. Bile acids: short and long term effects in the intestine. Scandinavian journal of gastroenterology. 2010;45:645–664. -PubMed
    1. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Frontiers in bioscience (Landmark edition) 2009;14:2584–2598. -PubMed
    1. Walters JR. Bile acid diarrhoea and FGF19: new views on diagnosis, pathogenesis and therapy. Nature reviews Gastroenterology & hepatology. 2014;11:426–434. -PubMed
    1. Edwards CA, Brown S, Baxter AJ, Bannister JJ, Read NW. Effect of bile acid on anorectal function in man. Gut. 1989;30:383–386. -PMC -PubMed
    1. Binder HJ, Rawlins CL. Effect of conjugated dihydroxy bile salts on electrolyte transport in rat colon. The Journal of clinical investigation. 1973;52:1460–1466. -PMC -PubMed

MeSH terms

Substances

Grants and funding

LinkOut - more resources