The Endocrine Disruptor Bisphenol A (BPA) Affects the Enteric Neurons Immunoreactive to Neuregulin 1 (NRG1) in the Enteric Nervous System of the Porcine Large Intestine.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

Influence of Acrylamide Administration on the Neurochemical Characteristics of Enteric Nervous System (ENS) Neurons in the Porcine Duodenum.

The digestive tract, especially the small intestine, is one of the main routes of acrylamide absorption and is therefore highly exposed to the toxic effect of acrylamide contained in food. The aim of this experiment was to elucidate the effect of low (tolerable daily intake-TDI) and high (ten times higher than TDI) doses of acrylamide on the neurochemical phenotype of duodenal enteric nervous system (ENS) neurons using the pig as an animal model. The experiment was performed on 15 immature gilts of the Danish Landrace assigned to three experimental groups: control (C) group-pigs administered empty gelatine capsules, low dose (LD) group-pigs administered capsules with acrylamide at the TDI dose (0.5 µg/kg body weight (b.w.)/day), and the high dose (HD) group-pigs administered capsules with acrylamide at a ten times higher dose than the TDI (5 µg/kg b.w./day) with a morning feeding for 4 weeks. Administration of acrylamide, even in a low (TDI) dose, led to an increase in the percentage of enteric neurons immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), neuronal nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VACHT) in the porcine duodenum. The severity of the changes clearly depended on the dose of acrylamide and the examined plexus. The obtained results suggest the participation of these neuroactive substances in acrylamide-inducted plasticity and the protection of ENS neurons, which may be an important line of defence from the harmful action of acrylamide.

Crosstalk between interleukin-6 and corticotropin-releasing factor modulate submucosal plexus activity and colonic secretion.

BACKGROUND: Irritable bowel syndrome (IBS) is a common disorder of the gut with symptoms such as diarrhoea, constipation, abdominal pain and bloating, that are frequently exacerbated by stress. Circulating levels of the pro-inflammatory cytokine, interleukin-6 (IL-6), which can activate colonic enteric neurons, are elevated in IBS patients. These studies aim to explore the relationship between IL-6 and the stress peptide, corticotropin-releasing factor (CRF) in colonic submucosal neurons. METHODS: Calcium imaging, Ussing chamber electrophysiology and immunohistochemistry were conducted on rat distal colons to investigate potential crosstalk between IL-6 and CRF. KEY RESULTS: Colonic secretions from the maternal separation rat model of IBS stimulated increases in intracellular calcium in naïve submucosal neurons via CRF1 receptors (n=15, p<0.05). Moreover, IL-6 (n=50, p<0.01) but not IL-1ß (n=46, p>0.05) or TNFα (n=46, p>0.05) potentiated the CRF-evoked calcium response. CRF (1µM, 1h, n=5) stimulation also induced colonic secretion of IL-6 and inhibited the pro-secretory effects of IL-6 on colonic ion transfer (n=12). CONCLUSIONS AND INFERENCES: These studies demonstrate the modulatory effects of CRF on colonic IL-6 secretion, neuronal activation and secretory function. These findings may provide an insight into the molecular mechanisms underlying symptom flares in IBS during periods of high stress.

Potentiation of mutant CFTR Cl- channel currents by the naturally occurring stilbene compound resveratrol.

Previously, we found that the naturally occurring stilbene compound resveratrol (RES) could potentiate cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. Because some wild-type CFTR activators also potentiate its mutant forms, we investigated effect of RES on the two most common forms of CF-related mutation (deltaF508 and G551D-CFTR). Cell-based fluorescence studies indicated that RES dose-dependently potentiated both deltaF508 and G551D mutant CFTR Cl- channel activities. Transepithelial Cl- currents were stimulated by RES in deltaF508 and G551D mutant CFTR-expressing FRT cells. Further excised inside-out patch-clamp measurements revealed that RES significantly induced the chloride current of deltaF508 and G551D mutant CFTRs by increasing the open time of the channels. In ex vivo studies, RES stimulated fluid secretion in mouse trachea by optical measurement of single gland secretion. These data suggested that RES is a potent deltaF508 and G551D mutant CFTR potentiator, and RES may present a novel class of therapeutic lead compounds in treating cystic fibrosis.

Relaxin exerts two opposite effects on mechanical activity and nitric oxide synthase expression in the mouse colon.

The hormone relaxin exerts a variety of functions on the smooth muscle of reproductive and nonreproductive organs, most of which occur through a nitric oxide (NO)-mediated mechanism. In the stomach and ileum, relaxin causes muscle relaxation by modulating the activity and expression of different nitric oxide synthase (NOS) isoforms region-dependently. Nothing is known on the effects of relaxin in the colon, the gut region expressing the highest number of neuronal (n) NOSß-immunoreactive neurons and mainly involved in motor symptoms of pregnancy and menstrual cycle. Therefore, we studied the effects of relaxin exposure in the mouse proximal colon in vitro evaluating muscle mechanical activity and NOS isoform expression. The functional experiments showed that relaxin decreases muscle tone and increases amplitude of spontaneous contractions; the immunohistochemical results showed that relaxin increases nNOSß and endothelial (e) NOS expression in the neurons and decreases nNOSα and eNOS expression in the smooth muscle cells (SMC). We hypothesized that, in the colon, relaxin primarily increases the activity and expression of nNOSß and eNOS in the neurons, causing a reduction of the muscle tone. The downregulation of nNOSα and eNOS expression in the SMC associated with increased muscle contractility could be the consequence of continuous exposue of these cells to the NO of neuronal origin. These findings may help to better understand the physiology of NO in the gastrointestinal tract and the role that the "relaxin-NO" system plays in motor disorders such as functional bowel disease.

Neuroprotective effect of quercetin on the duodenum enteric nervous system of streptozotocin-induced diabetic rats.

BACKGROUND: In diabetes mellitus (DM), hyperglycemia promotes changes in biochemical mechanisms that induce oxidative stress. Oxidative stress has been closely linked to adverse consequences that affect the function of the gastrointestinal tract caused by injuries to the enteric nervous system (ENS) that in turn cause neurodegeneration and enteric glial loss. Therapeutic approaches have shown that diet supplementation with antioxidants, such as quercetin, reduce oxidative stress. AIMS: This work sought to evaluate neurons and enteric glial cells in the myenteric and submucosal plexuses of the duodenum in diabetic rats supplemented with quercetin. METHODS: The duodenum of 24 rats, including a control group (C), control quercetin supplementation group (CQ), diabetic group (D), and diabetic quercetin supplementation group (DQ), were used to investigate whole mounts of muscular and submucosal layers subjected to immunohistochemistry to detect vasoactive intestinal peptide in the myenteric layer and double-staining for HuC-D/neuronal nitric oxide synthase (nNOS) and HuC-D/S100. RESULTS: A reduction of the general neuronal population (HuC/D) was found in the myenteric and submucosal plexuses (p < 0.001) in the D and DQ groups. The nitrergic subpopulation (nNOS) decreased only in the myenteric plexus (p < 0.001), and glial cells decreased in both plexuses (p < 0.001) in the D and DQ groups. In diabetic rats, quercetin supplementation reduced neuronal and glial loss. Diabetes promoted an increase in the cell body area of both the general and nitrergic populations. Quercetin supplementation only prevented neuronal hypertrophy in the general population. CONCLUSION: Supplementation with quercetin eased the damage caused by diabetes, promoting a neuroprotective effect and reducing enteric glial loss in the duodenum.

Macrolide antibiotics inhibit mucus secretion and calcium entry in Swine airway submucosal mucous gland cells.

Macrolide antibiotics such as erythromycin (EM) and azithromycin (AZM) are beneficial in the treatment of mucus hypersecretion in inflammatory pulmonary diseases. Several indirect and direct mechanisms of action have been proposed. This study investigates the direct effect of macrolides on secretory function of isolated submucosal mucous gland cells (SMGCs). We hypothesize that macrolides inhibit the calcium influx necessary for evoked mucus secretion. To test this, we quantified mucin protein release using enzyme-linked immunosorbent assay, calcium-activated K(+) (K(Ca)), and calcium-activated Cl(-) (Cl(Ca)) currents. We measured nonselective cation current (NSCC) using whole-cell patch-clamp techniques; intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 Ca(2+) imaging. We found that both EM and AZM are agonists at muscarinic receptors. EM (10 µM) evoked a small but significant increase in mucin release but inhibited the mucin release induced by subsequent acetylcholine (ACh) treatment. Both EM and AZM (10 µM) evoked K(Ca) and Cl(Ca) whole-cell currents, which were blocked by atropine. EM and AZM also accelerated the decay of inositol trisphosphate-induced K(Ca) and Cl(Ca) currents without changing the peak current amplitudes. Likewise, internal application of AZM (10 µM) enhanced the decay rate of ACh-induced K(Ca) and Cl(Ca) currents. EM (1-10 µM) and AZM (0.1-10 µM) slowly (over 25-30 min) inhibited thapsigargin (TG)-induced Ca(2+) entry when applied during the plateau phase of Ca(2+) entry but blunted TG-induced Ca(2+) entry by 70% after a 5-min pretreatment before initiating calcium entry. EM blocked TG-induced NSCC. We conclude that macrolide antibiotics are partial agonists at muscarinic receptors but inhibit stimulated mucus release by inhibiting calcium entry in SMGCs.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

BACKGROUND: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

5-HT(1A), SST(1), and SST(2) receptors mediate inhibitory postsynaptic potentials in the submucous plexus of the guinea pig ileum.

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.

Cholera toxin induces sustained hyperexcitability in submucosal secretomotor neurons in guinea pig jejunum.

BACKGROUND & AIMS: Neural mechanisms underlying cholera toxin (CT)-induced intestinal hypersecretion remain unclear. We investigated long-term excitability changes in vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) secretomotor neurons after prolonged luminal exposure to CT. METHODS: Isolated segments of guinea pig jejunum were incubated with saline or CT +/- neurotransmitter antagonist in the lumen; the submucosal plexus was then dissected clear, circumferentially adjacent to intact mucosa. Synaptic inputs and firing properties of S neurons in ganglia next to the mucosa in control saline were studied using intracellular recording. Neurons were processed for VIP and NPY immunoreactivity. RESULTS: Thirty S neurons (20 VIP(+), 7 NPY(+), 3 VIP(-)/NPY(-)) from CT-treated preparations and 27 control S neurons (19 VIP(+), 4 NPY(+), 4 VIP(-)/NPY(-)) in ganglia adjacent to intact mucosa were analyzed. VIP(+) and NPY(+) neurons in CT-treated preparations fired significantly more action potentials and for longer periods during injected depolarizing current pulses (50-350 pA) than control neurons. Addition of tetrodotoxin, hexamethonium, granisetron, or the neurokinin-1 (NK1) antagonist SR140333 during the CT incubation blocked CT-induced effects in both neuron types. The NK3 antagonist SR142801 blocked CT-induced effects in NPY(+) neurons and reduced the number of action potentials in VIP(+) neurons. Synaptic activity was unaffected by CT. CONCLUSIONS: CT induces specific and sustained hyperexcitability of secretomotor neurons in enteric pathways. CT acts in the mucosa. Its effect is neurally mediated and depends on 5-hydroxytryptamine-3, nicotinic, and NK1 receptors. This system represents a unique model to understand the neural mechanisms of action of CT and to identify therapeutic targets.

Effects and sites of action of a M1 receptor positive allosteric modulator on colonic motility in rats and dogs compared with 5-HT4 agonism and cholinesterase inhibition.

BACKGROUND: Muscarinic receptor 1 positive allosteric modulators (M1PAMs) enhance colonic propulsive contractions and defecation through the facilitation of M1 receptor (M1R)-mediated signaling. We examined M1R expression in the colons of 5 species and compared colonic propulsion and defecation caused by the M1PAM, T440, the 5-HT4 agonist, prucalopride, and the cholinesterase inhibitor, neostigmine, in rats and dogs. METHODS: M1R expression was profiled by immunostaining and in situ hybridization. In vivo studies utilized male SD rats and beagle dogs. Colonic propulsive contractions were recorded by manometry in anesthetized rats. Gut contractions in dogs were assessed using implanted force transducers in the ileum, proximal, mid, and distal colons. KEY RESULTS: M1R was localized to neurons of myenteric and submucosal plexuses and the epithelium of the human colon. A similar receptor localization was observed in rat, dog, mouse, and pig. T440 enhanced normal defecation in rats in a dose-dependent manner. Prucalopride also enhanced defecation in rats, but the maximum effect was half that of T440. Neostigmine and T440 were similarly effective in enhancing defecation, but the effective dose of neostigmine was close to its lethal dose. In rats, all 3 compounds induced colonic contractions, but the associated propulsion was strongest with T440. In dogs, intestinal contractions elicited by T440 propagated from ileum to distal colon. Prucalopride and neostigmine also induced intestinal contractions, but these were less well coordinated. No loss of effectiveness of T440 on defecation occurred after 5 days of repeated dosing. CONCLUSION AND INFERENCES: These results suggest that M1PAMs produce highly coordinated propagating contraction by actions on the enteric nervous system of the colon. The localization of M1R to enteric neurons in both animals and humans suggests that the M1PAM effects would be translatable to human. M1PAMs provide a potential novel therapeutic option for constipation disorders.

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa.

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.

Increased expression of CART, nNOS, VIP, PACAP, SP and GAL in enteric neurons of the porcine stomach prepyloric region following hydrochloric acid infusion.

INTRODUCTION: Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. MATERIAL AND METHODS: The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. RESULTS: A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. CONCLUSIONS: Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.

Activation of submucosal but not myenteric plexus of the gastrointestinal tract accompanies reduction of food intake by camostat.

UNLABELLED: It has been shown in the rat that endogenous cholecystokinin (CCK), released in response to the non-nutrient trypsin inhibitor camostat, reduces food intake at meals and increases Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation) in the dorsal vagal complex (DVC) of the hindbrain but not the myenteric plexus of the duodenum and jejunum. Experiment 1: We examined Fos-LI in the myenteric and the submucosal plexuses of the gut in response to orogastric gavage of camostat in rats. As we reported previously, camostat failed to increase Fos-LI in the myenteric plexus. We show here that camostat increased Fos-LI in the submucosal plexus of the duodenum and jejunum. Camostat also increased Fos-LI in the DVC. Experiment 2: Pretreatment with devazepide, a specific CCK(1) receptor antagonist abolished camostat-induced Fos-LI in the submucosal plexus and the DVC. Experiment 3: Bilateral subdiaphragmatic vagotomy reduced camostat-induced Fos-LI in the submucosal plexus approximately 40% and abolished it in the DVC. CONCLUSIONS: Activation of the submucosal plexus by cholecystokinin at the CCK(1) receptor accompanies stimulation of the dorsal vagal complex of the hindbrain and inhibition of food intake. Unlike the submucosal plexus, activation of the myenteric plexus is not necessary for cholecystokinin's influence on the dorsal vagal complex and food intake. The lack of activation in the myenteric plexus after camostat stimulation, in contrast to nutrient releasers of CCK such as oleate, suggests that intestinal stimulants can either release different amounts of CCK or cause release of CCK from I cells with different molecular forms of CCK. This would suggest that CCK-8 is released by camostat and is not able to travel to the myenteric plexus while a more stable form of CCK such as CCK-58 can travel to this site that is further away from the I cell.

Region-dependent effects of diabetes and insulin-replacement on neuronal nitric oxide synthase- and heme oxygenase-immunoreactive submucous neurons.

AIM: To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS: Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex- and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase (nNOS) and HuC/D, heme oxygenase (HO) 1 and peripherin, as well as HO2 and peripherin. The density of nNOS-, HO1- and HO2-immunoreactive (IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS: The total number of submucous neurons and the proportion of nNOS-, HO1- and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2- and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1- and HO2-IR submucous neurons was robust in the colon of controls (38.4%-50.8%), whereas it was significantly lower in the small intestinal segments (0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals. CONCLUSION: Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of nNOS-, HO1- and HO2-IR submucous neuronal density in the distal parts of the gut.

Fructose consumption impairs serotonergic signaling in the murine enteric nervous system.

The intake of free fructose has increased substantially since the development of high-fructose corn syrup. This has not only been associated with metabolic disorders but recent evidence also indicates that chronic fructose consumption can affect neuronal and cognitive function. In this study we investigated the effects of fructose consumption on serotonergic signaling and neuronal activity in the mouse submucous plexus. Male mice were put on a control or fructose (23% solution) diet for 6 weeks or were assigned to a recovery group that received normal water (2 weeks) after 4 weeks of fructose. At the end of the diet, gene expressions and enteric neuronal activity, after depolarization with high K(+) and 5-HT, were measured using Ca(2+) imaging and RT-qPCR, respectively. Even in the lack of gain weight and the absence of changes in duodenal permeability, the total number of 5-HT-responding neurons and the depolarization and 5-HT-evoked Ca(2+) amplitudes were significantly lower after fructose consumption. Expression of synaptobrevin CaV 2.1 and CaV 2.2 mRNA did not differ after fructose intake; however, CaV 2.1 mRNA levels were significantly higher in the recovery animals. SERT mRNA concentration, isolated from submucosal plexus containing mucosal epithelium, was significantly decreased after fructose consumption. Chronic fructose consumption impairs serotonergic signaling in the mouse submucous plexus, prior to weight gain and detectable intestinal permeability problems.

Effects of Oxaliplatin Treatment on the Enteric Glial Cells and Neurons in the Mouse Ileum.

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein-immunoreactive (IR) EGCs and protein gene product 9.5/ß-Tubulin III-IR neurons as well as increase in s100ß-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.

Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine.

The enteric nervous system contains intrinsic primary afferent neurons that allow mucosal stimulation to initiate reflexes without CNS input. We tested the hypothesis that submucosal primary afferent neurons are activated by 5-hydroxytryptamine (5-HT) released from the stimulated mucosa. Fast and/or slow EPSPs were recorded in submucosal neurons after the delivery of exogenous 5-HT, WAY100325 (a 5-HT(1P) agonist), mechanical, or electrical stimuli to the mucosa of myenteric plexus-free preparations (+/- extrinsic denervation). These events were responses of second-order cells to transmitters released by excited primary afferent neurons. After all stimuli, fast and slow EPSPs were abolished by a 5-HT(1P) antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, and by 1.0 microM tropisetron, but not by 5-HT(4)-selective antagonists (SB204070 and GR113808A) or 5-HT(3)-selective antagonists (ondansetron and 0.3 microM tropisetron). Fast EPSPs in second-order neurons were blocked by hexamethonium, and most slow EPSPs were blocked by an antagonist of human calcitonin gene-related peptide (hCGRP(8-37)). hCGRP(8-37) also inhibited the spread of excitation in the submucosal plexus, assessed by measuring the uptake of FM2-10 and induction of c-fos. In summary, data are consistent with the hypothesis that 5-HT from enterochromaffin cells in response to mucosal stimuli initiates reflexes by stimulating 5-HT(1P) receptors on submucosal primary afferent neurons. Second-order neurons respond to these cholinergic/CGRP-containing cells with nicotinic fast EPSPs and/or CGRP-mediated slow EPSPs. Slow EPSPs are necessary for excitation to spread within the submucosal plexus. Because some second-order neurons contain also CGRP, primary afferent neurons may be multifunctional and also serve as interneurons.

Expression of type 1 corticotropin-releasing factor receptor in the guinea pig enteric nervous system.

Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

Effects of ascorbic acid on the vasoactive intestinal peptide synthesis in the ileum submucous plexus of normal rats.

BACKGROUND: The aging process is a deteriorating process that attacks the gastrointestinal tract, causing changes in the number and size of neurons from the enteric nervous system. The activity of free radicals on enteric neurons is helped by the significant reduction of antioxidants. AIM: Evaluate the effect of the ascorbic acid supplementation on the neurons that produce the vasoactive intestinal peptide (VIP) in the submucous plexus of the ileum of normal rats for a period of 120 days. METHODS: Fifteen rats were divided in three groups: untreated control with 90 days, untreated control with 210 days and ascorbic acid-treated rats with 210 days. Ascorbic acid was given for 16 weeks from the 90th day of age by adding it to drinking water (1 g/L prepared fresh each day). The ileums were processed according to the immunohistochemistry technique for whole-mount preparation in order to detect the presence of VIP immunoreactive in the cellular bodies and nervous fibers in the neurons of the submucous plexus. We have verified their immunoreactivity and measured the cellular profile of 80 cellular bodies of VIP-ergic neurons from each studied group. RESULTS: The ascorbic acid supplementation did not alter physiological parameters such as water intake and food consumption of the three studied groups. We observed a significant increase of the cellular profile of VIP-ergic neurons in untreated control with 210 days when compared to untreated control with 90 days. The cellular profile of VIP-ergic neurons in ascorbic acid-treated rats with 210 days was bigger than those observed in others groups. CONCLUSION: The ascorbic acid had a neurotrophic effect on VIP-ergic neurons on the ileum after period 120 days of supplementation.