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Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2-6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2-4 dpi. PRRSV type 1 infection also induced dead cells (4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.

Hydroxyxanthones, the synthetic form of xanthones that predominantly found in mangosteen (Garcinia mangostana), have been indicated as a potent anti-inflammatory action. This study aimed to examine the action of various HDX forms in preventing a tight junction (TJ) barrier disruption induced by TNF-α or IL-1β through the inhibition of p-MLC expression in Caco-2 cell model. TJ barrier function was determined by transepithelial electrical resistance (TER) and flux measurement of fluorescein isothiocyanate-dextran (FD-4). 1-monoHDX or 1,3-diHDX (10 μM) promoted TER but not FD-4 permeability in the absence of cytokines. Pretreatment with 1-monoHDX at 10 μM for 24 h prevented TNF-α or IL-1β effect on TJ permeability. p-MLC protein expression by Western blot analysis, which was induced by IL-1β was inhibited with all HDXs. Tricyclic scaffold as the basic skeleton of all HDXs may be a potential candidate therapeutic for treatment of diseases-related leaky gut.

This study aimed to investigate whether endometrial tissues and endometrial epithelial cells were capable of secreting beta-defensin (BD)-1 and -2, and soy isoflavones genistein or daidzein could promote these BD secretions. The effect of genistein on Cl- secretion in correlation with the BD secretion was also examined.Endometrial tissues or glandular epithelial cell monolayer were mounted in Ussing chamber for measurement of electrical parameters. The sample solutions from apical and basolateral compartments were collected before and after genistein or daidzein addition for the measurement of BD-1 and-2 levels by using ELISA technique.Endometrial tissues and epithelial cells constitutively secreted both BD-1 and -2 mostly at the apical compartment. Both genistein and daidzein induced BDs secretion which reached a peak at 5-15 min. The apical secretion of BDs was coincidence with increased Cl- secretion induced by genistein.Endometrial tissues and epithelial cells contributes to basal host defense against infection by secretion of BD-1 and -2, which could be enhanced by genistein or daidzein. This finding implies that these potent constituents of soy isoflavones may be useful to promote the innate immune function of human endometrium.

The present study aimed to investigate the anti-inflammatory effect of phytoestrogen genistein (Ge) on the secretion of interleukin 6 (IL6) under lipopolysaccharide (LPS) stimulated conditions in human endometrial epithelial cell line RL95-2. The effects of Ge on expression of TLRs 2, 3, 4 and 9 proteins in response to the inflammatory development induced by LPS were also examined and compared with those of 17β-estradiol (E2).The RL95-2 cells were cultured in the estrogen-deprived media with or without bacterial endotoxin LPS 30 min prior to incubation with Ge (10-7, 10-6 or 10-5 M) or E2 (10-9 M) for 48 h. The culture media were collected at 1 and 24 h for IL6 measurement by the enzyme linked immunosorbent assay and the cell lysate for TLR protein expression analyzed by semi-quantitative Western blot.Ge or E2 did not alter the IL6 level in the absence of LPS; however, treatment with either Ge or E2 for 1 h up to 24 h decreased the IL6 level stimulated by LPS. The cells challenged with LPS significantly upregulated the TLRs 2 and 9 but suppressed the TLRs 3 and 4 protein expression. Forty eight h-treatment with Ge had no effect on the TLRs expression, whereas E2 down-regulated the increased TLR9 induced by LPS.Ge suppressed the inflammatory response by decreasing the IL6 level, but not associated with the alteration of TLRs-mediated pathway inducedby bacterial infection. In contrast, E2 decreased the IL6 secretion possibly due to the opposition on LPS-induced TLR9 expression. This provides the potential evidence of soy isoflavone genistein in alleviating the inflammation of endometrium following bacterial invasion.

Problem β-defensins are important innate chemical barriers that protect the endometrium from pathogen invasion. The effects of soy isoflavones, genistein and daidzein, on the expression and secretion of porcine β-defensins (PBD) in endometrial epithelial cells were investigated under normal or poly I:C-stimulated conditions. Method of study Primary cultured porcine endometrial epithelial (PE) cells were pretreated with genistein or daidzein followed by poly I:C inoculation. During treatment, the culture media were analyzed for PBD 1-4 secretion by ELISA and the total RNA for PBD gene expression by quantitative RT-PCR. Results Porcine endometrial epithelial cells constitutively expressed PBD 1-4 and secreted PBD-1, PBD-2, and PBD-4. Genistein and daidzein enhanced PBD-2 expression and PBD-2 and PBD-3 secretion. These compounds also potentiated PBD-2 and PBD-3 expression and secretion which were upregulated by poly I:C. Conclusion Soy isoflavones, genistein and daidzein, could be potentially used for promoting the innate host defense of endometrium against infection.

The present study aimed to investigate whether genistein, a potent phytoestrogen mainly found in soybean, modulated the expression of TLRs 2, 3, 4 and 9 proteins in human endometrial epithelial cell line RL95-2 under basal and polyinosinic-polycytidylic acid (poly I: C) stimulated conditions to mimic viral infection. The genistein effects were also compared with 17β-estradiol.The RL95-2 cells were cultured in the estrogen-deprived media with or without poly I: C 30 min prior to incubation with genistein (10(-7), 10(-6) or 10(-5) M) or 17β-estradiol (10(-9) M) for 48 h. The TLRs protein expression was analyzed by semi-quantitative Western blot.The cells expressed TLRs 3, 4 and 9 but a very low level of TLR2 proteins. Poly I: C significantly increased the TLRs 2 and 9 protein expressions whereas the TLRs 3 and 4 were reduced. Under basal condition, genistein at 10(-7) M increased the TLR2 while 17β-estradiol decreased the TLR4. All concentrations of genistein and 17β-estradiol attenuated the poly I: C induced increase in the TLR2. By contrast, both genistein at 10(-5) M and 17 β-estradiol further potentiated the TLR4 suppressed by poly I: C. Only 17β-estradiol was found to antagonize the poly I: C-induced changes in TLRs 3 and 9.Taken together, the present results that genistein increased the basal TLR2 and attenuated the viral component-induced TLR2 protein expression in human endometrial epithelial cells may indicate the potential role of this soy isoflavone in promoting the uterine immune function and probably alleviating the inflammation of endometrium following pathogen

The effect of prolactin (PRL) on ion transport across the porcine glandular endometrial epithelial cells was studied in primary cell culture using the short-circuit current technique. Addition of 1 μg/ml PRL either to the apical solution or to the basolateral solution produced a peak followed by a sustained increase in Isc, but with a lesser response when PRL was added apically. Basolateral addition of PRL increased the Isc in a concentration-dependent manner with a maximum effect at 1 μg/ml and an effective concentration value of 120 ng/ml. The PRL-stimulated Isc was significantly reduced by pretreatment with an apical addition of 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 μM), diphenylamine-2-carboxylic acid (1 mM) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (200 μM), Cl− channel blockers, but not by amiloride (10 μM), a Na+ channel blocker. In addition, pretreatment with bumetanide (200 μM), a Na+–K+–2Cl− cotransporter inhibitor, in the basolateral solution significantly reduced the PRL-stimulated Isc. Replacement of Cl− or in the bathing solutions also decreased the Isc response to PRL. Pretreatment of the monolayer with AG490 (50 μM), an inhibitor of JAK2 activity significantly inhibited the PRL-induced increase in Isc. Western blot analysis of the porcine endometrial epithelial cells revealed the presence of short isoform of PRL receptor (PRLR-S) that could be regulated by 17β-estradiol. The results of this investigation showed that PRL acutely stimulated anion secretion across the porcine endometrial epithelial cells possibly through PRLR-S present in both apical and basolateral membranes. The PRL response appeared to be mediated by the JAK2-dependent pathway.

Contamination with bacterial endotoxin causes the disruption of the tight junction (TJ) barrier. We investigated the ameliorative effect of dietary flavonoids genistein (Ge) and daidzein (Di) in normal or lipopolysaccharide (LPS)-induced disruption of epithelial barrier function of the endometrium. Using the immortalized porcine glandular endometrial epithelial cells (PEG), transepithelial electrical resistance (TER) and FITC-dextran flux (FD-4) across the monolayer were measured. The mRNA expression of TJ proteins, zona occludens-1 (ZO1), and claudin-1, -3, -4, -7 and -8 was evaluated by real-time RT-PCR for coinciding effect of Ge or Di occurred at the gene transcription level. The results revealed that Ge and Di altered the TER, depending on times and concentrations. Low concentration (10−10 M) of both compounds decreased the TER, whereas higher concentrations (10−8and 10−6 M) increased the TER which was not related to the FD-4 flux. The increased TER by Ge or Di was parallel to the induction ofclaudin-3and-4or-8mRNA expression respectively. With LPS inoculation, all isoflavone treatments inhibited the decreased TER induced by LPS, but only Ge (10−8or 10−6 M) or Di (10−10or 10−6 M) was coincidence with the decreased FD-4 flux. Under this LPS-stimulated condition, some or all examined TJ gene expressions appeared to be promoted by specific concentration of Ge or Di respectively. Our findings suggest that the soy isoflavones treatment could promote and restore the impaired endometrial barrier function caused by LPS contamination.

Anxiety is a symptom reflecting the dysregulation of monoaminergic neurotransmitters which may be modulated by estrogen. In our current study, we investigated the effects of chronic estrogen administration (10 microg/kg, s.c. for 4 weeks) on anxiety-like behavior using the elevated plus-maze with the corresponding changes of monoamines in the brain regions contributing to anxiety. The behavioral test revealed that estrogen-treated rats (Ovx+E(2)) spent more time in the open arm of the maze as well as a higher time/entry ratio in open arms than ovariectomized (Ovx) rats, indicating an anxiolytic property of estrogen. The increase in open arm time corresponded to an increase in uterine weight, indicated a correlation between the function of estrogen and its anxiolytic effect. Measurements of brain monoamines following estrogen treatment revealed decreases in norepinephrine, dopamine and serotonin in all of the brain regions studied, which also lead to an increase in turnover rates. The concentrations of norepinephrine in caudate putamen, of dopamine in nucleus accumbens, of serotonin in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and substantia nigra and of the serotonin metabolite, the 5-hydroxyindolacetic acid in substantia nigra of Ovx+E(2) rats were significantly lower than those of Ovx rats. Interestingly, the uterine weight was negatively correlated with the changes of dopamine and serotonin (with the exception of the hippocampus), suggesting a regulatory role of estrogen on these systems. From these data, we concluded that, in fact, there is a relationship between estrogen and monoamines (i.e. serotonin, dopamine) in modulating the anxiety-like behaviors in female rats.

The present study aimed to investigate the purgative effects of barakol, the purified extract of Cassia siamea Lam., on the longitudinal smooth muscle contractions of the rat ileum. The extract increased the force of spontaneous muscle contractions in a concentration-dependent manner (EC50=0.3 mM). Saxitoxin (0.3 microM) abolished the stimulatory effects of barakol, a result indicating a neural mechanism of action. In addition, atropine (10 microM) but not propanolol (10 microM) or phentolamine (10 microM), partially inhibited barakol-induced smooth muscle contractions suggesting that cholinergic nerves were involved. The motor effects of barakol were further examined in muscle strips treated with catecholamines to suppress spontaneous contractile activity and decrease muscle tone. Norepinephrine or dopamine (10 microM) decreased the amplitude of spontaneous contractions by 72% and 18%, respectively. Pretreatment of the tissues with barakol (1 mM) significantly decreased the inhibitory effect of norepinephrine by 60%, but not that of dopamine. Its ability to potentiate atropine- and saxitoxin-sensitive contractions and inhibit the antimotility actions of norepinephrine suggests that barakol may increase longitudinal smooth muscle contractions by decreasing the inhibitory effect of norepinephrine on excitatory cholinergic motor neurons. Barakol may produce a purgative action in small intestine which may be clinically important in patients with intestinal hypomotility disorders.

Genistein (Ge) and daidzein (Di), the active isoflavones primarily found in soybean, have various beneficial health effects. The present study aims to investigate the effects of these isoflavones on tight junction (TJ) barrier integrity in immortalized endometrial epithelial cells (IMM). Expressions of the TJ integrity markers, zona occludens-1 (ZO-1) and claudins (cldn)-1, -3, -4, -7 and -8 were analyzed by quantitative reverse transcription and real-time polymerase chain reaction (RT-PCR) and transepithelial electrical resistance (TER) by voltohmmeter. Treatment with Ge (10 nM or 1 μM) for 48 h increased the TER, and this was correlated with the mRNA expression of cldn-3 and/or ZO-1. However, low dose of Ge (0.1 nM) decreased both the TER and the expression of cldn-3 and/or ZO-1. Di did not alter the expression of the TJ proteins. Our results suggest that genistein increases barrier function possibly via the expression of cldn-3 and ZO-1. This represents an important protective role of genistein against...

β-defensins (BD) are antimicrobial peptides that contribute the mucosal innate immunity. Its expression is regulated by sex steroid hormones. Phytoestrogens genistein (Ge) and daidzein (Di) mostly ...

Immediate hypersensitivity reactions in the intestinal mucosa evoke active chloride secretion which enhances the elimination of luminal antigens. The prosecretory actions of histamine and other soluble mediators of anaphylaxis are mediated by submucosal neurons, as are the antisecretory actions of opioid antidiarrheal medications. We tested the hypothesis that the selective δ-opioid receptor agonist [ d -Pen2, d -Pen5]enkephalin (DPDPE) alters anaphylaxis-associated ileal anion secretion in vitro. Sheets of ileal mucosa with attached submucosa from guinea pigs sensitized to cow's milk were mounted in Ussing chambers under short-circuit conditions. Mucosal sheets responded to the serosal application of the milk protein, β-lactoglobulin, with a rapid rise in transepithelial short-circuit current (Isc); in contrast, the egg protein, ovalbumin, was without effect. Pretreatment of tissues with the neuronal conduction blocker, saxitoxin, or the H1 histamine receptor antagonist, diphenhydramine, but not the opioid receptor antagonist, naloxone, significantly reduced mucosal responses to antigen. [ d -Pen2, d -Pen5]enkephalin (0.1 μM, serosal addition) decreased baseline Isc, but potentiated mucosal responses to antigen; its effects were abolished in tissues pretreated with naloxone. These results suggest that immediate hypersensitivity reactions in the guinea pig ileal mucosa are mediated by submucosal neural circuits that are phasically modulated by both mast cell products and opioids.

Opiates have potent antidiarrheal actions thatare mediated in part by delta-opioid receptors (DOR). Weexamined DOR localization within subregions of porcineileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR wasobtained after reverse transcription-polymerase chainreaction cloning of forebrain RNA; it encoded the end oftransmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93%nucleotide identity with human DOR. Positive signals forDOR mRNA were found in all subregions of the porcineileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-likeimmunoreactivity was localized in neurons withinmyenteric and submucous ganglia, longitudinal andcircular smooth muscle, and villous lamina propria. TheDOR agonist [D-Ser2, Leu5, Thr6]enkephalin(DSLET) attenuated circular smooth muscle contractionsin porcine ileum that were evoked by electricalstimulation of myenteric cholinergic neurons. Theseresults are consistent with previous reports of the DOR-mediatedneuromodulation that underlies the antipropulsive andantisecretory effects of opioids in the intestinaltract.

Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (Isc) which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl- current and barium-sensitive basolateral K+ current while inhibiting the apical K+ current. Genistein failed to increase the Isc in the presence of forskolin or IBMX, but did increase the Isc in UTP. Pretreatment with genistein also abolished the increase in the Isc when induced by forskolin, IBMX or UTP. However, Ca2+-chelating BAPTA-AM did not affect the genistein-induced increase in the Isc. The genistein-stimulated Isc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia.

Cytochrome c (CytC) released from mitochondria induces apoptosis in both normal and tumor cells. Expression of Fas ligand (FasL) helps maintain tumor cell survival by inducing apoptosis of Fas-bearing anti-tumor immune cells. A risk of endometrial cancer has been reported to associate with phytoestrogen consumption. Therefore the effects of phytoestrogens, genistein and daidzein, on FasL and CytC protein expression were examined in primary cultured porcine endometrial cells (PE) and human cancerous endometrial cells (RL95-2) by Western blot analysis. Both cells were cultured in standard medium (SM) and switched to estrogen-deprived medium (SF) with or without 17beta-estradiol (E, 1 nM), genistein (10 microM) or daidzein (10 microM) for 48 h. FasL (25 kDa) which was found only in RL95-2 cells was upregulated in SF compared to SM. Treatment of RL95-2 cells with E, daidzein or genistein significantly increased the FasL expression by 7-10 folds. In the present study, low level of CytC was detected in both cells cultured in SM but markedly increased in SF by 1.5-2 folds. The SF-induced increase in CytC level was reversed by genistein or daidzein while E suppressed CytC in PE cells, but not in RL95-2 cells. The findings suggest that genistein and daidzein appear to act as a survival factor by inhibiting intracellular apoptogenic initiator in both normal and cancer endometrial cells. In addition, estrogen and phytoestrogens inducing the death signal FasL expressed by cancerous endometrial cells may cause the tumor progression. Thus, consuming phytoestrogen as a supplement should be awareness in patient with endometrial cancer.

The effects of estrogen on anxiety-like behaviors have been widely studied but the mechanisms responsible are still inconclusive. The purpose of the current study was to compare the effects of transient high levels of endogenous estrogen and chronic exogenous estrogen treatment on the anxiety-like behaviors using the elevated T-maze (ETM) test. In addition, serotonin (5-HT) and its metabolite (5-HIAA), serotonin reuptake transporter (SERT) and tryptophan hydroxylase enzyme (TPH) were measured at the end of the study and correlated to the task performances. Female sham-operated rats in proestrous phase (Sham-Pro) and ovariectomized rats treated with or without 17beta-estradiol (10 microg/kg, s.c.; Ovx+E(2) or Ovx) for 4 weeks were used. In the ETM test, the Ovx+E(2) group had reduced inhibitory avoidance responses compared to others, suggesting that exogenous E(2) replacement is anxiolytic, while escape latency was prolonged in the Sham-Pro group suggesting endogenous E(2) is panicolytic. Further, the serotonin turnover rate (5-HIAA/5-HT ratio) in the hippocampus and nucleus accumbens was highest in the Ovx+E(2) group. While the TPH protein in the midbrain of Ovx rats was significantly higher than others, the SERT levels were not significantly different among groups in all measured brain areas. In conclusion, Ovx rats with chronic estrogen administration and Sham-Pro rats with naturally high levels of estrogen, demonstrated anxiolytic behavior by exhibiting different forms of anxiety that related to the changes in the function of serotonergic system.

The regulation of prolactin (PRL) on electrolyte transport was investigated in primary culture of porcine glandular endometrial epithelial cells using Ussing chamber technique. PRL produced a peak ...

Barakol is a purified extract of Cassia siamea, a plant that has been used as a laxative in traditional medicine. In this study, the effect of barakol on anion transport across the rat colon epithelium was investigated. Colonic epithelium was mounted in Ussing chambers and bathed with Ringer's solution. Addition of 1 mM barakol to the basolateral solution produced a slow increase in short-circuit current (Isc) in proximal colon and distal colon by 24.5 +/- 2.2 and 24.2 +/- 1.4 microA/cm(2), respectively. Barakol increased Isc in a concentration-dependent manner with an EC(50) value of 0.4 mM. The barakol-stimulated increase in Isc was inhibited by subsequent treatment with 500 microM diphenylamine-2-carboxylic acid or 400 microM glibenclamide added to the apical solution and 200 microM bumetanide added to the basolateral solution. Pretreatment of the tissues with 200 microM bumetanide, but not 10 microM amiloride, completely abolished the barakol-increased Isc. Ion substitution experiments showed an inhibition of barakol-stimulated Isc in chloride-free solution but not in bicarbonate-free solution. In addition, pretreatment of tissues with 10 microM tetrodotoxin or 10 microM indomethacin, but not 1 microM atropine or 10 microM hexamethonium, partially inhibited the Isc response by barakol. The present results demonstrated the stimulatory effect of barakol on the bumetanide-sensitive chloride secretion in rat colon. The effect of barakol was partly mediated by the stimulation of submucosal nerves and through the release of cyclooxygenase metabolites. These findings thus provide an explanation for the underlying mechanism of barakol as a secretagogue in mammalian colon.

Histamine and the mast cell degranulator, compound 48/80 produced elevations in short-circuit current, an electrical measure of active anion secretion, across porcine ileal mucosa sheets mounted in Ussing chambers. Luminally-applied beta-lactoglobulin produced similar effects in mucosal sheets from cow's milk-sensitized pigs. Their secretory effects were attenuated by blockers of H(1)-histamine receptors, neuronal conduction or epithelial Na(+)/K(+)/Cl(-) cotransport. The delta-opioid agonist [D-Pen(2), D-Pen(5)]enkephalin suppressed mucosal responses to these substances in a naltrindole-reversible manner. Furthermore, submucosal mast cells and delta-opioid receptor-immunoreactive nerve fibers were observed in close juxtaposition. Intestinal neural pathways linking immediate hypersensitivity to secretory host defense appear to express inhibitory delta-opioid receptors.

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.