A Technique of Measurement of Gastrointestinal Luminal Nutrient Sensing and These Absorptions: Ussing Chamber (Short-Circuit Current) Technique.

The gastrointestinal (GI) tract is a series of hollow organs that play roles in food digestion and nutrient absorption. To perform these functions, they should recognize the luminal environment and elicit adequate physiological responses, including digestive juice secretion, peristaltic movements, etc. The Ussing chamber technique is an electrophysiological method for measuring transepithelial ion transport and permeability as short-circuit current (Isc) and transepithelial electrical tissue conductance (Gt) or resistance (TEER), respectively, in vitro. This technique can be applied for the measurement of luminal nutrient sensing and absorption. This article introduces practical methods for measuring luminal nutrient sensing and absorption using intestinal mucosa specimens isolated from humans and experimental animals.

Role of PGE2 in colonic motility: PGE2 attenuates spontaneous contractions of circular smooth muscle via EP4 receptors in the rat colon.

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called 'giant contractions' (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.

The effects of viable and non-viable Lactobacillus gasseri CP2305 cells on colonic ion transport and corticotropin releasing factor-induced diarrhea.

The effect of non-viable lactic acid bacteria on gastrointestinal physiology and dysfunction remains still unclear. Previous clinical trials have reported that Lactobacillus gasseri CP2305 (CP2305) exerts stress-relieving and anti-flatulent effects regardless of cell viability. In this study, we investigated the effect of viable and non-viable CP2305 cells on electrical field stimulation (EFS)-evoked increases in short-circuit current (Isc) using the Ussing chamber technique. In mucosal-submucosal preparations of rats, both viable and non-viable CP2305 cells significantly and acutely inhibited the EFS-evoked increases in Isc in the middle and distal colon and rectum but not in proximal colon. The inhibition of EFS-evoked Isc differed from strain to strain. Peripheral injection of corticotropin releasing factor (CRF) is known to mimic diarrhea symptoms in rats. Therefore, we examined the chronic effects of CP2305 cells on CRF-induced diarrhea in the rat model. Treatment with viable and non-viable CP2305 cells significantly improved CRF-induced diarrhea in the rat model. However, the treatment did not affect the fecal pellet output. These findings suggest that CP2305 has an important role in gastrointestinal physiology and dysfunction.

Gut microbiota confers host resistance to obesity by metabolizing dietary polyunsaturated fatty acids.

Gut microbiota mediates the effects of diet, thereby modifying host metabolism and the incidence of metabolic disorders. Increased consumption of omega-6 polyunsaturated fatty acid (PUFA) that is abundant in Western diet contributes to obesity and related diseases. Although gut-microbiota-related metabolic pathways of dietary PUFAs were recently elucidated, the effects on host physiological function remain unclear. Here, we demonstrate that gut microbiota confers host resistance to high-fat diet (HFD)-induced obesity by modulating dietary PUFAs metabolism. Supplementation of 10-hydroxy-cis-12-octadecenoic acid (HYA), an initial linoleic acid-related gut-microbial metabolite, attenuates HFD-induced obesity in mice without eliciting arachidonic acid-mediated adipose inflammation and by improving metabolic condition via free fatty acid receptors. Moreover, Lactobacillus-colonized mice show similar effects with elevated HYA levels. Our findings illustrate the interplay between gut microbiota and host energy metabolism via the metabolites of dietary omega-6-FAs thereby shedding light on the prevention and treatment of metabolic disorders by targeting gut microbial metabolites.

Effects of an Enteral Formula Containing Fermented Dairy Products on Epithelial Ion Transport in Rat Intestines.

Diarrhea is the most common complication of enteral nutrition (EN). Pro/prebiotics are typically used to prevent diarrhea during EN. This study aimed to demonstrate the effects of enteral formula containing fermented dairy products (FDPs) and galacto-oligosaccharides on intestinal mucosal functions in rats. After feeding rats with regular rodent chow (RRC), standard formula (STD-F), and FDP-containing formula (FDP-F) for 2 wk, the rats were sacrificed with their intestines removed. Then, the electrophysiological properties of intestinal epithelia were measured using the Ussing chamber. In addition, organic acids and microbiota in the cecal contents were analyzed. In FDP-F-fed rats, electrical nerve activation-evoked increase in short-circuit current (Isc) in the cecum and middle colon was reduced compared with STD-F-fed rats. Mucosal propionate-evoked changes in Isc in FDP-F-fed rats were also reduced in the terminal ileum. The total cecal organic acid concentration in STD-F-fed rats decreased compared with RRC-fed rats, and approximately half was recovered in FDP-F-fed rats, which contributed to the recovery of acetate and butyrate concentrations. In microbiota analysis, the density of total bacteria, particularly Bifidobacterium, in cecal contents increased in FDP-F-fed rats. In conclusion, the consumption of FDP-F changed the total amounts and components of gut microbiota and organic acids, and resulted in inhibitory changes in mucosal luminal stimulant- and nervous system-mediated fluid secretory function. These findings suggest that FDP-F might prevent the incidence of diarrhea during EN.

Activation of muscarinic receptors prevents TNF-α-mediated intestinal epithelial barrier disruption through p38 MAPK.

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.

Diversity of the intestinal microbiota differently affects non-neuronal and atropine-sensitive ileal contractile responses to short-chain fatty acids in mice.

Non-neuronal and atropine-sensitive ileal contractile responses to short chain fatty acids (SCFAs) are detected in the neonatal stage, and change with age or inflammatory conditions. However, the roles of luminal SCFAs in developmental changes have not yet been elucidated. We examined ileal contractile responses to SCFAs in mice colonized with different SCFA-producing intestinal microbiota under normal and inflammatory conditions. Using conventional (Conv), germ-free (GF), and gnotobiotic mice infected with Bifidobacterium (GB-bif), Propionibacterium (GB-prop), or Lactobacillus (GB-lact), ileal contractions were measured in 1-day-old neonates and 7-week-old mice using an isotonic transducer. Contractions occurred in all 1-day-old neonates, and were significantly desensitized in the adult stage in the Conv, GB-bif, and GB-prop groups, but not in the GF and GB-lact groups. An injection of lipopolysaccharide frequently restored desensitized contractions; however, the contraction rate did not change in the GF and GB-lact groups. The relative mRNA expression of a SCFA receptor (GPR43) or nicotinic acetylcholine receptor α7 was weaker in the GF group (0.3-fold or 0.4-fold expression level, respectively) than in the Conv group. In conclusion, the luminal inhabitation of SCFA-producing bacteria may potentiate the regulation of non-neuronal and atropine-sensitive ileal contractile responses to SCFAs under healthy and inflammatory conditions.

Effects of ε-viniferin, a dehydrodimer of resveratrol, on transepithelial active ion transport and ion permeability in the rat small and large intestinal mucosa.

ε-Viniferin is a dehydrodimer of resveratrol, a polyphenol synthesized in many plants, including grapevine. The present study investigated the effects of ε-viniferin and resveratrol on epithelial secretory and barrier functions in isolated rat small and large intestinal mucosa. Mucosa-submucosa tissue preparations of various segments of the rat large and small intestines were mounted on Ussing chambers, and short-circuit current (Isc) and tissue conductance (Gt) were continuously measured. The mucosal addition of ε-viniferin (>10(-5) mol/L) and resveratrol (>10(-4) mol/L) to the cecal mucosa, which was the most sensitive region, induced an increase in Isc and a rapid phase decrease (P-1) followed by rapid (P-2) and broad (P-3) peak increases in Gt in concentration-dependent manners. Mucosal ε-viniferin (10(-4) mol/L), but not resveratrol (10(-4) mol/L), increased the permeability of FITC-conjugated dextran (4 kDa). The mucosal ε-viniferin-evoked changes in Isc (Cl(-) secretion), but not in Gt, were attenuated by a selective cyclooxygenase (COX)-1 inhibitor and a selective EP4 prostaglandin receptor. The mucosal ε-viniferin-evoked increase in Isc was partially attenuated, and P-2, but not P-1 or P-3, change in Gt was abolished by a transient receptor potential cation channel, subfamily A, member 1 (TRPA1) inhibitor. Moreover, the mucosal ε-viniferin concentration-dependently attenuated the mucosal propionate (1 mmol/L)-evoked increases in Isc and Gt Immunohistochemical studies revealed COX-1-immunoreactive epithelial cells in the cecal crypt. The present study showed that mucosal ε-viniferin modulated transepithelial ion transport and permeability, possibly by activating sensory epithelial cells expressing COX-1 and TRPA1. Moreover, mucosal ε-viniferin decreased mucosal sensitivity to other luminal molecules such as short-chain fatty acids. In conclusion, these results suggest that ε-viniferin modifies intestinal mucosal transport and barrier functions.

Desacetyl bisacodyl-induced epithelial Cl(-) secretion in rat colon and rectum.

The purpose of this study was to clarify the mode of desacetyl bisacodyl (DAB)-induced secretory action in intestinal tissues using an Ussing chamber assay. DAB is the active metabolite of the laxative bisacodyl. In mucosal-submucosal preparations, mucosal application of DAB induced a transient decrease followed by subsequent increases in short-circuit current and tissue conductance in a concentration-dependent manner. DAB-induced responses occurred from the middle colon to the rectal segment but not in the proximal colon. Moreover, these responses were not observed under chloride (Cl(-))-free conditions or in the presence of DAB on the serosal side of the mucosalsubmucosal specimens. Treatment with tetrodotoxin had no effect on the DAB-induced responses; however, mucosal treatment with a COX inhibitor piroxicam resulted in the elimination of responses. These results suggest that DAB may contribute to the laxative action by inducing Cl(-) secretion which is associated with the COX signaling pathway. This study also demonstrated that the DAB target molecule is present on the mucosal side from the middle colon to the rectal segment.

Activation of muscarinic cholinoceptor ameliorates tumor necrosis factor-α-induced barrier dysfunction in intestinal epithelial cells.

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR.

Luminal trypsin induces enteric nerve-mediated anion secretion in the mouse cecum.

Proteases play a diverse role in health and disease. An excessive concentration of proteases has been found in the feces of patients with inflammatory bowel disease or irritable bowel syndrome and been implicated in the pathogenesis of such disorders. This study examined the effect of the serine protease, trypsin, on intestinal epithelial anion secretion when added to the luminal side. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I sc) was measured. Trypsin added to the mucosal (luminal) side increased I sc with an ED50 value of approximately 10 µM. This I sc increase was suppressed by removing Cl(-) from the bathing solution. The I sc increase induced by 10-100 µM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by a neurokinin-1 receptor antagonist, but not by a muscarinic or nicotinic ACh-receptor antagonist. The trypsin-induced I sc increase was also significantly inhibited by a 5-hydroxytryptamine-3 receptor (5-HT3) antagonist and substantially suppressed by the simultaneous addition of both 5-HT3 and 5-HT4 receptor antagonists. We conclude that luminal trypsin activates the enteric reflex to induce anion secretion, 5-HT and substance P playing important mediating roles in this secreto-motor reflex. Luminal proteases may contribute to the cause of diarrhea occurring with some intestinal disorders.

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 host's energy metabolism via GLP-1 release, as well as the mucosal defense system.

Role of PGE2 in the colonic motility: PGE2 generates and enhances spontaneous contractions of longitudinal smooth muscle in the rat colon.

The aim of this study was to determine which PGE2 receptors (EP1-4 receptors) influence colonic motility. Mucosa-free longitudinal smooth muscle strips of the rat middle colon spontaneously induced frequent phasic contractions (giant contractions, GCs) in vitro, and the GCs were almost completely abolished by a cyclooxygenase inhibitor, piroxicam, and by an EP3 receptor antagonist, ONO-AE3-240, but enhanced by tetrodotoxin (TTX). In the presence of piroxicam, exogenous PGE2, both ONO-AE-248 (EP3 agonist), and ONO-DI-004 (EP1 agonist) induced GC-like contractions, and increased the frequency and amplitude. These effects of EP receptor agonists were insensitive to TTX and ω-conotoxins. In immunohistochemistry, the EP1 and EP3 receptors were expressed in the longitudinal smooth muscle cells. These results suggest that the endogenous PGE2 spontaneously generates and enhances the frequent phasic contractions directly activating the EP1 and EP3 receptors expressed on longitudinal smooth muscle cells in the rat middle colon.

Taste sensing in the colon.

The colonic lumen is continually exposed to many compounds, including beneficial and harmful compounds that are produced by colonic microflora. The intestinal epithelia form a barrier between the internal and luminal (external) environments. Chemical receptors that sense the luminal environment are thought to play important roles as sensors and as modulators of epithelial cell functions. The recent molecular identification of various membrane receptor proteins has revealed the sensory role of intestinal epithelial cells. Nutrient sensing by these receptors in the small intestine is implicated in nutrient absorption and metabolism. However, little is known about the physiological roles of chemosensors in the large intestine. Since 1980s, researchers have examined the effects of short-chain fatty acids (SCFA), the primary products of commensal bacteria, on gut motility, secretion, and incretin release, for example. In this decade, the SCFA receptor genes and their expression were identified in the mammalian colon. Furthermore, many other chemical receptors, including taste and olfactory receptors have been found in colonic epithelial cells. These findings indicate that the large intestinal epithelia express chemosensors that detect the luminal contents, particularly bacterial metabolites, and induce the host defense systems and the modulation of systemic metabolism via incretin release. In this review, we describe the local effects of chemical stimuli on the lumen associated with the expression pattern of sensory receptors. We propose that sensory receptors expressed in the colonic mucosa play important roles in luminal chemosensing to maintain homeostasis.

Chemical synthesis and formulation design of a PEGylated vasoactive intestinal peptide derivative with improved metabolic stability.

The present study aimed to design a PEGylated VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), with improved metabolic stability, and develop its respirable powder (RP) formulation for inhalation therapy. IK312532 was chemically conjugated with PEG (5 kDa, P5K), the physicochemical and biochemical properties of which were characterized by CD spectral analysis, binding assays, and metabolic stability. CD spectral analysis demonstrated that PEG conjugation had no impact on the conformational structure of IK312532. Although the receptor-binding activity of IK312532/P5K (IC50: 82 nM) was estimated to be ca. 30-fold less than that of IK312532 (IC50: 2.8 nM), the metabolic stability of IK312532/P5K was highly improved. The IK312532/P5K was jet-milled and blended with lactose carrier particles to provide RP formulation of IK312532/P5K (IK312532/P5K-RP). In vitro inhalation performance and in vivo pharmacological effects of the IK312532/P5K-RP in antigen-sensitized rats were also evaluated. In cascade impactor analyses, fine particle fraction of IK312532/P5K-RP was calculated to be ca. 37%. Insufflation of IK312532/P5K-RP (150 µg of IK312532/P5K) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decreases in inflammatory biomarkers and granulocyte recruitment in pulmonary tissue 24h after the antigen challenge. From these findings, PEGylation of a VIP derivative, as well as its strategic application to the RP formulation, may be a viable approach to improve its therapeutic potential for the treatment of airway inflammatory diseases.

Inhalable sustained-release formulation of long-acting vasoactive intestinal peptide derivative alleviates acute airway inflammation.

The present study was undertaken to develop a respirable sustained-release powder (RP) formulation of long-acting VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), using PLGA nanospheres (NS) with the aim of improving the duration of action. NS formulation of IK312532 (IK312532/NS) was prepared by an emulsion solvent diffusion method in oil, and a mixture of the IK312532/NS and erythritol was jet-milled and mixed with lactose carrier to obtain the IK312532/NS-RP. Physicochemical properties were characterized focusing on appearance, particle size, and drug release, and in vivo pharmacological effects were assessed in antigen-sensitized rats. The IK312532/NS with a diameter of 140 nm showed a biphasic release pattern in distilled water with ca. 20% initial burst for 30 min and a sustained slow release up to ca. 55% for 24h. Laser diffraction analysis demonstrated that IK312532/NS-RP had fine dispersibility and suitable particle size for inhalation. In antigen-sensitized rats, insufflated IK312532/NS-RP (10 µg of IK312532/rat) could suppress increases of granulocyte recruitment and myeloperoxidase in pulmonary tissue for up to 24h after antigen challenge, although IK312532-RP at the same dose was less effective with limited duration of action. From these findings, newly prepared IK312532/NS-RP might be of clinical importance in improving duration of action and medication compliance for treatment of airway inflammatory diseases.

Subepithelial trypsin induces enteric nerve-mediated anion secretion by activating proteinase-activated receptor 1 in the mouse cecum.

Serine proteases are versatile signaling molecules and often exert this function by activating the proteinase-activated receptors (PAR(1)-PAR(4)). Our previous study on the mouse cecum has shown that the PAR(1)-activating peptide (AP) and PAR(2)-AP both induced electrogenic anion secretion. This secretion mediated by PAR(1) probably occurred by activating the receptor on the submucosal secretomotor neurons, while PAR(2)-mediated anion secretion probably occurred by activating the receptor on the epithelial cells. This present study was aimed at using trypsin to further elucidate the roles of serine proteases and PARs in regulating intestinal anion secretion. A mucosal-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I(sc)) was measured. Trypsin added to the serosal side increased I(sc) with an ED(50) value of approximately 100 nM. This I(sc) increase was suppressed by removing Cl(-) from the bathing solution. The I(sc) increase induced by 100 nM trypsin was substantially suppressed by tetrodotoxin, and partially inhibited by an NK(1) receptor antagonist, by a muscarinic Ach-receptor antagonist, and by 5-hydroxytryptamine-3 (5-HT(3)) and 5-HT(4) receptor antagonists. The I(sc) increase induced by trypsin was partially suppressed when the tissue had been pretreated with PAR(1)-AP, but not by a pretreatment with PAR(2)-AP. These results suggest that the serine protease, trypsin, induced anion secretion by activating the enteric secretomotor nerves. This response was initiated in part by activating PAR(1) on the enteric nerves. Serine proteases and PARs are likely to be responsible for the diarrhea occurring under intestinal inflammatory conditions.

Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon.

In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10(-6)-10(-3) M) induced Cl(-) and HCO(3)(-) secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E(2); however, this secretion was not affected by TTX, atropine, or extracellular Ca(2+) depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP(4) receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP(4) receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

Chemosense for luminal environment in the large intestine.

Gut lumen is continually exposed to many agents, including noxious compounds. The intestinal epithelia form a barrier between the internal and luminal (external) environments. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and as modulators of epithelial cell functions. The Molecular analysis of various epithelial cell membrane receptor proteins has elucidated the sensory role of these cells in the gut chemosensing system. Nutrient sensing systems by these receptors in the small intestinal epithelia are thought to influence nutrient metabolism and local physiological function. Much less is known, however, about the physiological roles of chemosensing in the large intestine. We have investigated the contractile and secretory effects of short-chain fatty acids (SCFAs), the primary products of commensal bacteria, and the expression of SCFA receptors in the large intestine. The findings indicate that the epithelia in the large intestine also detect and respond to luminal contents, particularly bacterial metabolites, for host defense. We recently reported that luminal bitter tastants and odorants affect transepithelial ion transport in human and rat colon, and that putative receptors are expressed in colonic mucosa. In this review, we describe the secretory effects of chemical stimuli on lumen associated with the expression pattern of sensory receptors, focusing on the large intestine.

Effects of luminal thymol on epithelial transport in human and rat colon.

Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca(2+)-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.