Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon

Abstract

Key points: Luminal short-chain fatty acids (SCFAs) influence gut physiological function via SCFA receptors and transporters. The contribution of an SCFA receptor, free fatty acid receptor (FFA)3, to the enteric nervous system is unknown. FFA3 is expressed in enteric cholinergic neurons. Activation of neural FFA3 suppresses Cl(-) secretion induced by nicotinic ACh receptor activation via a Gi/o pathway. Neural FFA3 may have an anti-secretory function by modulating cholinergic neural reflexes in the enteric nervous system.

Abstract: The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl(-) secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13 μm. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the Gi/o pathway. Luminal propionate induced Cl(-) secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

Figure 1. Antibody characterization

A and B, frozen sections of CSMG were double‐stained with antibodies to FFA3 (A) (green) and PGP9.5 (B) (red). C, merged image showing FFA3 and PGP9.5 colocalization. D, preabsorption of FFA3 antibody in the section of CSMG. E, liver section as a negative control had no FFA3‐IR. Nuclei were counterstained with DAPI (blue). F–H, FFA3‐IR (red) in rat FFA3‐ (F), rat FFA2‐ (G) or mock‐(H) transfected HeLa cells. After a 48 h culture, the transfected cells were fixed by 4% paraformaldehyde for 15 min and reacted with FFA3 antibody. I, FFA3 mRNA expression assessed by real‐time RT‐PCR in the CSMG, in the muscle layer of proximal colon (PCs) and in the liver (L). *P < 0.05. J, western blot using FFA3 antibody (1 μg ml⁻¹) detected a ∼38 kDa band (arrow) in the extracted protein from rat colonic submucosa (SM) and absorbed by antigen peptide (10 μg ml⁻¹). M, standard proteins with molecular size (kDa) on the left. K and L, immunohistochemistry with α‐CGRP antibody in serial sections of rat dorsal root ganglia. α‐CGRP‐IR (arrow, K) was absorbed by antigen peptide (5 μg ml⁻¹, L).

Figure 2. Localization of FFA3‐IR in rat proximal colon

A–C, frozen sections were double‐stained with FFA3 (A) and with PGP9.5 (B). The merged image (C) showed that FFA3 colocalized with PGP9.5 in subepithelial nerve fibres (solid arrowheads), in the submucosal plexus (SMP), in intramuscular nerves in circular (CM) and longitudinal (LM) muscle, and in myenteric plexus (MP). Scattered FFA3‐positive epithelial cells had morphology resembling enteroendocrine cells (open arrowhead). D, preabsorption abolished FFA3‐IR. Nuclei were counterstained with DAPI (blue). E–G, whole mounts of mucosa (E), submucosa (F) and myenteric plexus (G) of proximal colon were stained with FFA3 antibody (red).

Figure 3. Localization of FFA3‐IR in cholinergic nerves in whole mounts of mucosa and in submucosal plexus

A–C, FFA3 (A) and VAChT (B) in mucosal plexus. Both FFA3‐ and VAChT‐IR nerves (merged image in C) surrounded the crypts. D–F, FFA3 (D) and CHT1 (E) expression in the submucosal plexus. Most FFA3‐IR nerve fibres and endings expressed CHT1 (arrows in F), whereas FFA3‐IR was faint in CHT1‐IR neuron somata (asterisks). G, CHT1 (red) and VIP (green) expressed in mucosal plexus. VIP‐IR and CHT1‐IR were detected in individual nerve fibres. H and I, FFA3 and α‐CGRP (H) or calbindin (I) in submucosal plexus. Calbindin‐IR neuron (asterisk) had no FFA3‐IR.

Figure 4. Effect of FFA3 ligands on CCh‐induced I_sc increases in Ussing‐chambered rat proximal colon

Preparations were incubated with inhibitors during the stabilization period, whereas MQC or AR420626 was added 15 min before CCh application. All chemicals were added into the serosal bath. A, representative I _sc traces in mucosa–submucosa preparations in response to CCh (10 μm) in the presence or absence of MQC. B, concentration‐dependent inhibition of MQC on CCh‐evoked ΔI _sc in mucosa–submucosa preparations. A single concentration of MQC was applied to each preparation. C, CCh‐evoked ΔI _sc in the presence or absence of MQC (100 μm), AR420626 (10 μm), TTX (1 μm), hexamethonium (Hex: 10 μm) or atropine (Atr: 10 μm) in mucosa–submucosa preparations. *P < 0.05 vs. vehicle. D, effect of bethanechol (100 μm) or nicotine (100 μm) on I _sc in the presence or absence of MQC (100 μm) in mucosa–submucosa preparations. *P < 0.05 vs. vehicle. E, effect of MQC and TTX (1 μm) on CCh‐evoked ΔI _sc in mucosal preparations. F, effect of MQC in the presence or absence of PTX (500 ng ml⁻¹) in mucosa–submucosa preparations. *P < 0.05. G and H, effect of NaP, NaAc (1 mm) or 3‐chloropropionate (3Cl‐P, 1 mm) on CCh‐evoked ΔI _sc in mucosa–submucosa preparations. *P < 0.05 vs. vehicle. n.s., not significant. SCFA solutions were adjusted to pH 7.4. I, effect of CF3‐MQC with or without MQC on CCh‐evoked ΔI _sc in mucosa–submucosa preparations. A single concentration of CF3‐MQC was applied to each preparation. MQC (30 μm) was added 10 min after the application of CF3‐MQC (100 μm).

Figure 5. Effect of MQC on NaP‐ or FK‐evoked ΔI_sc

A, representative I _sc traces in response to luminal NaP (5 mm) in the presence (solid triangles) or absence (open circles) of TTX (1 μm). B, luminal NaP‐evoked ΔI _sc in the presence or absence of TTX (1 μm), hexamethonium (100 μm) or atropine (10 μm), or in the submucosa‐stripped mucosal preparations (Mucosa). C, representative I _sc traces in mucosa–submucosa preparations. NaP (5 mm) was added into the luminal bath following luminal DMSO (grey) or MQC (1 mm, black). MQC decreased basal I _sc, as did DMSO (vehicle) and inhibited the response to NaP. D, NaP‐evoked ΔI _sc in the presence or absence of MQC in the serosal (s) or luminal (m) bath 10 min before the application of NaP (5 mm). *P < 0.05 vs. vehicle. E, FK (5 μm) was added into the serosal bath and ΔI _sc was measured in the presence or absence of individual concentrations of serosal MQC.