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.

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.

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.

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.

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.

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.

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.

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.

Architecture and Chemical Coding of the Inner and Outer Submucous Plexus in the Colon of Piglets.

In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.

Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X1, P2X2, P2X3 channels, P2Y and A3 metabotropic receptors in neurotransmission.

RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

By what mechanism does ondansetron inhibit colonic migrating motor complexes: does it require endogenous serotonin in the gut wall?

BACKGROUND: 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5-HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT-depleted preparation. METHODS: Mice were injected with reserpine, 24 h prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT. KEY RESULTS: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was not different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally or significantly more effective at inhibiting CMMC frequency compared with control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs. CONCLUSIONS & INFERENCES: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus or 5-HT in myenteric neurons.

Ginger and its pungent constituents non-competitively inhibit activation of human recombinant and native 5-HT3 receptors of enteric neurons.

BACKGROUND: Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome. METHODS: Heterologously expressed human 5-HT3 A or 5-HT3 AB receptors were characterized by means of Ca(2+) influx studies using HEK293 cells. Complementing Ca(2+) measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds. KEY RESULTS: We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [(3) H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3 A and 5-HT3 AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons. CONCLUSIONS & INFERENCES: Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.

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.

Submucous rather than myenteric neurons are activated by mucosal biopsy supernatants from irritable bowel syndrome patients.

BACKGROUND: We previously showed that colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS) activate neurons of the human submucous plexus, an area with densely packed immune cells. Based on the concept that mucosa-nerve signaling is altered in IBS, we tested in this study whether the nerve sensitizing effect of IBS mucosal biopsy supernatants is more prominent in the submucous than myenteric plexus. METHODS: Fast neuroimaging with the voltage-sensitive dye Di-8-ANEPPS was used to record activity of guinea-pig submucous and myenteric neurons after application of constipation (C)- and diarrhea (D)-IBS supernatants (three each) and four supernatants from healthy control subjects. Results are based on recordings from 4731 neurons. KEY RESULTS: Control supernatants did not evoke significant responses in submucous or myenteric neurons. In contrast, all IBS supernatants evoked a significant spike discharge (median 3.6 Hz) in 46% of submucous neurons. This activation was significantly stronger than in the myenteric plexus where even twice the amount of supernatants evoked a lower spike frequency (median 2.1Hz) in only 8.5% of neurons. Pharmacological studies revealed serotonin, histamine, and proteases as components mediating neuronal activation. Individual application of these components revealed that only serotonin evoked a significantly stronger activation of submucous compared with myenteric neurons. CONCLUSIONS & INFERENCES: Direct neuronal activation by IBS mucosal biopsy supernatants is primarily a feature of submucous rather than myenteric neurons. This is associated with a stronger excitation of submucous neurons by serotonin. The plexus-specific effects support the concept that altered mucosa-nerve signaling underlies disturbances in IBS.

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.

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.

Leptin excites enteric neurons of guinea-pig submucous and myenteric plexus.

BACKGROUND: Leptin, one of the most prominent mediators released from adipocytes, influences neuronal activity in the central nervous system. The enteric nervous system (ENS) expresses leptin receptors but consequence of activation of these receptors on enteric neuron activity has not been systematically studied. An adipocyte-ENS axis is suggested by close apposition between enteric nerves and adipocytes. The aim of this study was to investigate the effects of leptin on guinea-pig submucous and myenteric neurons. METHODS: Using voltage sensitive dye imaging, we recorded neural responses to application of leptin (0.0625 nmol L(-1)) in myenteric and submucous neurons, nicotine (10 µmol L(-1)) served as a reference for neuronal excitation. Mucosal ion secretion and muscle activity were measured in vitro with Ussing and organ bath techniques, respectively. KEY RESULTS: Leptin induced spike discharge in 13.6% of submucous neurons and in 8.2% of myenteric neurons (1.1 ± 0.9 and 1.2 ± 1.0 Hz, respectively). Although there was an overlap of nicotine and leptin responses, 38.5% of submucous and 25% of myenteric neurons activated by leptin did not respond to nicotine. Leptin did not inhibit ongoing spike discharge or fast excitatory postsynaptic potentials. Leptin (0.0625 nmol L(-1)) did not affect mucosal secretion or muscle activity suggesting a subtle modulatory action of leptin at the level of the ENS. CONCLUSIONS & INFERENCES: Leptin activates submucous and myenteric neurons indicating relevance for adipocyte-ENS signaling. These results set the basis for further studies to reveal the functional correlate of the neural action of leptin in the ENS.

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.