Atsukazu Kuwahara

Short-chain fatty acid receptor, GPR43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are the major anions in the large intestinal lumen. They are produced from dietary fiber by bacterial fermentation and are known to have a variety of physiological and pathophysiological effects on the intestine. In the present study, we investigated the expression of the SCFA receptor, GPR43, in the rat distal ileum and colon. Expression of GPR43 was detected by reverse transcriptase/polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. mRNA for GPR43 was detected, by RT-PCR, in extracts of the whole wall and separated mucosa from the ileum and colon and from muscle plus submucosa from the ileum, but not from muscle plus submucosa preparations from the colon. We raised a rabbit antiserum against a synthesized fragment of rat GPR43; this was specific for rat GPR43. GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extracts. By immunohistochemistry, GPR43 immunoreactivity was localized to enteroendocrine cells expressing peptide YY (PYY), whereas 5-hydroxytryptamine (5-HT)-immunoreactive (IR) enteroendocrine cells were not immunoreactive for GPR43. Mast cells of the lamina propria expressing 5-HT were also GPR43-IR. The results of the present study suggest that the PYY-containing enteroendocrine cells and 5-HT-containing mucosal mast cells sense SCFAs via the GPR43 receptor. This is consistent with physiological data showing that SCFAs stimulate the release of PYY and 5-HT from the ileum and colon.

Expression of the short-chain fatty acid receptor, GPR43, in the human colon

Short-chain fatty acids (SCFAs), 2–4 carbon monocarboxylates including acetate, propionate and butyrate, are known to have a variety of physiological and pathophysiological effects on the intestine. Previously, we reported that the SCFA receptor, G-protein coupled receptor 43 (GPR43), is expressed by enteroendocrine and mucosal mast cells in the rat intestine. In the present study, expression and localization of GPR43 were investigated in the human large intestine. Gene and protein expression of GPR43 in the human ascending colon was analyzed by reverse transcriptase/polymerase chain reaction and Western blotting, respectively. In addition, localization of GPR43 was investigated by immunohistochemistry. In RT-PCR analysis, GPR43 mRNA was detected in whole wall mRNA samples. Western blotting analysis revealed the expression of GPR43 protein in whole wall and scraped mucosa protein samples, but not in muscle or submucosa. GPR43 immunoreactivity was observed in the intracellularly in enterocytes and in the peptide YY-immunoreactive enteroendocrine cells. These results indicate that the short chain fatty acid receptor, GPR43 is expressed by enteroendocrine L cells containing peptide YY in the human large intestine.

Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure

Abstract Since definitive morphological studies showing the luminal release of serotonin have not been reported, we used a perfused system which allows physiological monitoring and biochemical as well as morphological evidence indicating release of serotonin from enterochromaffin cells. Isolated vascularly and luminally perfused rat duodenums exposed to 5–35 cmH2O of luminal pressure were measured for release of serotonin into the blood vessels and intestinal lumen. Immediately after raising the luminal pressure, the duodenum was fixed for immunoelectron microscopic localization of serotonin. Peristaltic contraction and serotonin content of the perfusates were continuously measured. The luminal release of serotonin increased with elevated intraluminal pressure, but the vascular release of serotonin was not altered. Tetrodotoxin had no effect on the pressure-stimulated luminal serotonin release. Enterochromaffin cells in control animals without increased luminal pressure contained immunogold-labeled secretory granules in the apical and basal cytoplasm. After intraluminal pressure increased, many apical secretory granules were no longer dense and immunogold particles were localized over the cytoplasmic matrix and microvilli. These findings indicate that luminal serotonin release is increased after raising the intraluminal pressure and serotonin, normally stored in the secretory granules of enterochromaffin cells, appears to be released into the cytoplasmic matrix and then diffuses or is transported into the intestinal lumen.

Secretory effects of a luminal bitter tastant and expressions of bitter taste receptors, T2Rs, in the human and rat large intestine.

Taste transduction molecules, such as Galpha(gust), and taste receptor families for bitter [taste receptor type 2 (T2R)], sweet, and umami, have previously been identified in taste buds and the gastrointestinal (GI) tract; however, their physiological functions in GI tissues are still unclear. Here, we investigated the physiological function and expression of T2R in human and rat large intestine using various physiological and molecular biological techniques. To study the physiological function of T2R, the effect of a bitter compound, 6-n-propyl-2-thiouracil (6-PTU), on transepithelial ion transport was investigated using the Ussing chamber technique. In mucosal-submucosal preparations, mucosal 6-PTU evoked Cl(-) and HCO(3)(-) secretions in a concentration-dependent manner. In rat middle colon, levels of 6-PTU-evoked anion secretion were higher than in distal colon, but there was no such difference in human large intestine. The response to 6-PTU was greatly reduced by piroxicam, but not by tetrodotoxin. Additionally, prostaglandin E(2) concentration-dependently potentiated the response to 6-PTU. Transcripts of multiple T2Rs (putative 6-PTU receptors) were detected in both human and rat colonic mucosa by RT-PCR. In conclusion, these results suggest that the T2R ligand, 6-PTU, evokes anion secretion, and such response is regulated by prostaglandins. This luminal bitter sensing mechanism may be important for host defense in the GI tract.

Short-chain fatty acid receptor and its contribution to glucagon-like peptide-1 release.

Background: Gut microbiota affects host homeostasis and dysbiosis causes host diseases. Therefore, uncovering the sensing mechanism of bacterial metabolites such as short-chain fatty acid (SCFA) may help us to understand the host-microbiota interaction both in physiological and nonphysiological conditions. Summary: The colonic lumen is continually exposed to many kinds of chemicals, including beneficial and harmful compounds that are produced by gut microbiota in addition to ingested nutrients. In the mammalian colon SCFAs such as acetate, propionate and butyrate are produced by bacterial fermentation and reach about 100 mM under physiological conditions. In this decade, SCFA receptor genes and their expression in the intestine have been identified as free fatty acid receptor (FFA)2 and FFA3. The FFAs are located in colonic enteroendocrine L cells producing and releasing an insulinotropic hormone, glucagon-like peptide-1 (GLP-1), and an anorectic hormone, peptide YY. Recent in vivo and in vitro studies suggest that SCFAs stimulate gut hormone secretion. Therefore, the SCFA-FFA signal is likely to be important for gut physiological functions. Key Message: Colonic epithelial cells express chemical receptors that detect the luminal contents, particularly bacterial metabolites, and may be involved in the hosts energy metabolism via GLP-1 release, as well as the mucosal defense system.

Neural and non-neural mediation of propionate-induced contractile responses in the rat distal colon

Abstract  Short‐chain fatty acids (SCFAs), including propionate, butyrate and acetate, are fermentation products of carbohydrates in the colon. We investigated the contractile effects of SCFAs on the rat distal colon. Mechanical activity of the circular muscle in strip preparations was recorded in vitro. Propionate and butyrate concentration‐dependently (10 μmol L−1–10 mmol L−1) induced rapid, large amplitude phasic contractions (the first phase) followed by tonic contractions (the second phase). Acetate itself had no effect on muscle activity, although preincubation with acetate attenuated both phases of the propionate‐induced response. The propionate‐induced phasic contraction was attenuated by atropine, tetrodotoxin and the 5‐HT4 receptor antagonist SB‐204070. The propionate‐induced tonic contraction was attenuated by the cyclo‐oxygenase inhibitor piroxicam. Antagonists of 5‐HT1A, 5‐HT2A and 5‐HT3 receptors had no effect on the responses. Propionate‐induced responses were not observed in mucosa‐free preparations. These results suggest that propionate acts on receptors in the mucosa causing the release of 5‐HT from enterochromaffin cells. 5‐HT acts through 5‐HT4 receptors on the endings of intrinsic primary afferent neurones that in turn activate cholinergic motor neurones that contract the circular muscle. Propionate also causes tonic contraction, via prostaglandin release, in the rat distal colon.

Regulation of intestinal secretion involved in the interaction between neurotransmitters and prostaglandin E2

In this short review, it will be described that neurotransmitter‐induced secretion in the intestine may be influenced by the tissue level of prostaglandin E2 (PGE2). In the normal condition, vasoactive intestinal polypeptide (VIP) and acetylcholine (ACh) are the predominant neurotransmitters of secretomotor neurones. VIP and ACh activate distinct second messenger systems in epithelial cells, i.e. adenosine 3′, 5′‐cyclic monophosphate (cAMP) and calcium ion (Ca2+), respectively. An increase in intracellular cAMP induces a small amount of chloride (Cl–) secretion in epithelial cells, while simultaneous increases in intracellular Ca2+ and cAMP greatly enhances the cAMP‐induced Cl– secretion. When the concentration of prostaglandins reaches a high level in the intestinal tissue substance P, which is a neurotransmitter of sensory neurones, can also induce a massive Cl– secretion by cross‐potentiation of cAMP and Ca2+ in epithelial cells. In conclusion, it is considered that the concentration of tissue PGE2 may indicate tissue alert level, and when this level elevates, PGE2 enhances ACh and SP‐induced Cl– secretion, thus mediating massive fluid secretion for host defence.

5-HT activates nitric oxide-generating neurons to stimulate chloride secretion in guinea pig distal colon

The participation of nitric oxide (NO) in serotonin (5-hydroxytryptamine; 5-HT)-evoked chloride secretion in guinea pig distal colon was examined. Submucosal/mucosal segments were mounted in Ussing flux chambers, and an increase in short-circuit current ( I sc) was used as an index of secretion. Addition of 5-HT to the serosal side produced a concentration-dependent (10-7-10-5M) increase in I sc caused by chloride secretion. N G-nitro-l-arginine (l-NNA) significantly reduced the 5-HT-evoked early (P-1) and late (P-2) responses to 61.1 and 70.6% of control, respectively. Neurally evoked response was also inhibited by l-NNA. The NO donor sodium nitroprusside (SNP, 10-4 M) increased basal I sc mainly because of chloride secretion. The SNP-evoked response was significantly reduced by tetrodotoxin but was unchanged by atropine or indomethacin. These results suggest that the 5-HT-evoked increase in I sc is associated with an NO-generating mechanism. Atropine significantly reduced the 5-HT (10-5 M)-evoked P-1 and P-2 responses to 71.8 and 19.7% of control, respectively. Simultaneous application of atropine andl-NNA further decreased the 5-HT-evoked responses more than either drug alone; application ofl-NNA and atropine decreased the 5-HT-evoked P-1 and P-2 responses to 68.5 and 39.2% of atropine-treated tissues, respectively. These results suggest that noncholinergic components of P-1 and P-2 responses are 71.8 and 19.7% of control, respectively, and that NO components of P-1 and P-2 responses are 32 and 61%, respectively, of the noncholinergic component of the 5-HT-evoked responses. The results provide evidence that NO may participate as a noncholinergic mediator of 5-HT-evoked chloride secretion in guinea pig distal colon.The participation of nitric oxide (NO) in serotonin (5-hydroxytryptamine; 5-HT)-evoked chloride secretion in guinea pig distal colon was examined. Submucosal/mucosal segments were mounted in Ussing flux chambers, and an increase in short-circuit current (Isc) was used as an index of secretion. Addition of 5-HT to the serosal side produced a concentration-dependent (10(-7)-10(-5) M) increase in Isc caused by chloride secretion. NG-nitro-L-arginine (L-NNA) significantly reduced the 5-HT-evoked early (P-1) and late (P-2) responses to 61.1 and 70.6% of control, respectively. Neurally evoked response was also inhibited by L-NNA. The NO donor sodium nitroprusside (SNP, 10(-4) M) increased basal Isc mainly because of chloride secretion. The SNP-evoked response was significantly reduced by tetrodotoxin but was unchanged by atropine or indomethacin. These results suggest that the 5-HT-evoked increase in Isc is associated with an NO-generating mechanism. Atropine significantly reduced the 5-HT (10(-5) M)-evoked P-1 and P-2 responses to 71.8 and 19.7% of control, respectively. Simultaneous application of atropine and L-NNA further decreased the 5-HT-evoked responses more than either drug alone; application of L-NNA and atropine decreased the 5-HT-evoked P-1 and P-2 responses to 68.5 and 39.2% of atropine-treated tissues, respectively. These results suggest that noncholinergic components of P-1 and P-2 responses are 71.8 and 19.7% of control, respectively, and that NO components of P-1 and P-2 responses are 32 and 61%, respectively, of the noncholinergic component of the 5-HT-evoked responses. The results provide evidence that NO may participate as a noncholinergic mediator of 5-HT-evoked chloride secretion in guinea pig distal colon.

Is nitric oxide involved in 5-HT-induced fluid secretion in the gut?

The present study was undertaken to test the hypothesis that 5-HT stimulates nitric oxide (NO) generating neurons, and that these neurons participate in the mediation of 5-HT-induced fluid secretion. 5-HT induced electrogenic Cl- secretion in guinea-pig distal colon. This response was abolished by tetrodotoxin but not by atropine. The maximum response to 5-HT (10(-5) M) was inhibited by approximately 65% (P < 0.05, n = 6) by the NO synthase inhibitor, NG-nitro-L-arginine (L-NNA, 10(-4) M). The substrate of NO synthase, L-Arg (10(-3) M) reversed the inhibition of 5-HT-induced secretions by L-NNA. 5-HT-induced diarrhea in fasted mice was reduced by atropine in vivo. NG-Nitro-L-Arg methyl ester (L-NAME, 1-32 mg/kg, i.p.) dose-dependently inhibited 5-HT (1 mg/kg)-induced diarrhea. The inhibitory effect of L-NAME was reversed by L-Arg, but not D-Arg (600 mg/kg, i.p., respectively). Taken together, these data suggest that 5-HT-induced fluid secretion in the gut is partly due to the activation of neurons that generate NO.

Galanin suppresses neurally evoked contractions of circular muscle in the guinea-pig ileum

The effect of galanin, a 29 amino-acid peptide, on intestinal smooth muscles was studied in guinea-pig ileum. Galanin did not affect the basal activity of longitudinal or circular muscles. Galanin decreased neurally evoked circular muscle contractions in a dose-dependent manner, but failed to affect neurally evoked longitudinal muscle contractions. Galanin also decreased neurally evoked circular muscle contractions in the presence of atropine. The neurally evoked phasic contractions were blocked by TTX. These findings indicate that galanin has an inhibitory role in circular muscle contractions via myenteric neurons in the guinea-pig ileum.