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BACKGROUND Neurorehabilitation strategies for chronic poststroke (PS) oropharyngeal dysphagia (OD) have been mainly focused on the neurostimulation of the pharyngeal motor cortex with only marginal effects. In contrast, treatments targeting the PS oropharyngeal sensory pathway dysfunction offer very promising results, but there is little knowledge on the underlying mechanisms. We aimed to explore the neurophysiological mechanisms behind the effect of three sensory neurostimulation strategies. METHODS We carried out a randomized two-blinded parallel group's crossover sham-controlled clinical trial in 36 patients with unilateral stroke and chronic unsafe swallow to investigate the effect of repetitive transcranial magnetic stimulation (rTMS) of the primary sensory cortex (A), oral capsaicin (B) and intra-pharyngeal electrical stimulation (IPES; C). The effect was evaluated immediately after the interventions with videofluoroscopy (VFS) and motor/sensory evoked potentials (MEP/SEP). KEY RESULTS Interventions induced no changes in the biomechanics of the swallow response during VFS. However, an enhancement of motor cortex excitability (latency shortening and increased size of thenar MEP) was found with active interventions (A + B + C, and B/C alone; P

Background Oropharyngeal dysphagia (OD) treatment is moving away from compensatory strategies toward active treatments that improve swallowing function. The aim of this study was to assess the acute therapeutic effect of TRPA1/M8 agonists in improving swallowing function in OD patients. Methods Fifty-eight patients with OD caused by aging, stroke, or neurodegenerative disease were included in a three-arm, quadruple-blind, randomized clinical trial (NCT02193438). Swallowing safety and efficacy and the kinematics of the swallow response were assessed by videofluoroscopy (VFS) during the swallow of 182 ± 2 mPa·s viscosity (nectar) boluses of a xanthan gum thickener supplemented with (a) 756.6 μmol/L cinnamaldehyde and 70 μmol/L zinc (CIN-Zn) (TRPA1 agonists), (b) 1.6 mmol/L citral (CIT) (TRPA1 agonist), or (c) 1.6 mmol/L citral and 1.3 mmol/L isopulegol (CIT-ISO) (TRPA1 and TRPM8 agonists). The effects on pharyngeal event-related potentials (ERP) were assessed by electroencephalography. Key results TRPA1 stimulation with either CIN-Zn or CIT reduced time to laryngeal vestibule closure (CIN-Zn P = .002, CIT P = .023) and upper esophageal sphincter opening (CIN-Zn P = .007, CIT P = .035). In addition, CIN-Zn reduced the penetration-aspiration scale score (P = .009), increased the prevalence of safe swallows (P = .041), and reduced the latency of the P2 peak of the ERP. CIT-ISO had no positive effect on biomechanics or neurophysiology. No significant adverse events were observed. Conclusions and inferences TRPA1 stimulation with CIN-Zn or CIT improves the swallow response which, in the case of CIN-Zn, is associated with a significant improvement in cortical activation and safety of swallow. These results provide the basis for the development of new active treatments for OD using TRPA1 agonists.

BACKGROUND: Previous studies have found that TRPV1 and TRPA1 receptor agonists improve swallow response in patients with oropharyngeal dysphagia (OD), but little is known about the expression of these receptors in the human oropharynx. The aim of this study was to assess the expression and localization of TRPV1 and TRPA1 in human samples from the oropharynx of healthy patients, to provide the basis for new pharmacological treatments for OD. METHODS: Samples from oropharyngeal regions innervated by cranial nerves V, IX, and X (tongue, pharynx, and epiglottis) were obtained during ENT surgery and processed either for mRNA (21 patients) or for immunohistochemical assays (seven patients). The expression analysis was performed with RT-qPCR using ACTBh as reference gene. Hemotoxylin and eosin staining was used to study the histology; the immunohistochemical assay used (i) neuron-specific enolase to detect nerve fibers or (ii) fluorescent probes to locate TRPV1 and TRPA1. RESULTS: TRPV1 was expressed in the three studied regions, with higher levels in CN V region (tongue) than in CN X region (epiglottis; p < 0.05), and was localized at epithelial cells and nociceptive fibers in all studied regions. TRPA1 was also expressed in all studied regions, but was always localized below the basal lamina. No immunoreactivity for TRPA1 was found on epithelial cells. CONCLUSIONS & INFERENCES: TRPV1 and TRPA1 are widely expressed in the human oropharynx with two distinct patterns. Our study further confirms that TRPV1/A1 receptors are promising therapeutic targets to develop active treatments for OD patients. ispartof: Neurogastroenterology and Motility vol:28 issue:1 pages:91-100 ispartof: location:England status: published

Background Colonic samples from asymptomatic diverticulosis (DS) patients presented enhanced electrical field stimulation (EFS)-contractions, in an earlier study of ours, suggesting increased endogenous responses. The aim of this study was to explore changes in excitatory neuromuscular transmission and to assess the pharmacodynamics of spasmolytic agents in DS. Methods Circular muscle strips from sigmoid colon of DS patients (n = 30; 69.5 ± 14.8 years) and controls (n = 32; 64.7 ± 16.2 years) were studied using organ baths to evaluate the direct effect of excitatory agonists (carbachol, neurokinin A [NKA] and substance P [SP]), and the effect of antagonists (atropine and NK2 antagonist GR94800) and spasmolytic drugs (otilonium bromide [OB] and N-butyl-hyoscine) on the contractions induced by EFS-stimulation of excitatory motorneurons. qRT-PCR was also performed to compare mRNA expression of M2, M3, NK2 receptors and L-type calcium channels. Key Results Contractions to carbachol (Emax: 663.7 ± 305.6% control vs 2698.0 ± 439.5% DS; p

Background Neuro-transmission impairment could be associated to motility changes observed in patients with diverticular disease. Therefore, the objective was to characterize the inhibitory neuro-muscular transmission and gene expression changes of the enteric inhibitory pathways in patients with diverticulosis (DS). Methods Circular muscle strips from sigmoid colon of patients with DS and controls were studied using the organ bath technique to evaluate spontaneous contractility and enteric motor neurons stimulated by electrical field and qRT-PCR to assess the expression of nNOS, iNOS, P2Y1R and PGP9.5. Key Results Patients with DS presented decreased spontaneous rhythmic contractions (SRC) that were significantly enhanced after incubation with L-NNA (1 mM) and TTX (1 μM), and unaffected by the P2Y1 antagonist MRS2500 (1 μM). Stimulation on enteric motor neurons caused an increased duration of the latency of OFF-contractions in DS group (p

Oropharyngeal Dysphagia (OD) is both underestimated and underdiagnosed as a cause of malnutrition and respiratory complications following stroke. OD occurs in more than 50% of stroke patients. Aspiration pneumonia (AP) occurs in up to 20% of acute stroke patients and is a major cause of mortality after discharge. Systematic screening for OD should be performed on every patient with stroke before starting oral feeding, followed, if appropriate by clinical and instrumental (videofluroscopy and/or fiberoptic endoscopy) assessment. Bolus modification with adaptation of texture and viscosity of solids and fluids and postural adjustments should be part of the minimal treatment protocol, but they do not change the impaired swallow physiology nor promote recovery of damaged neural swallow networks in stroke patients. To this purpose, two new neurostimulation approaches are being developed to stimulate cortical neuroplasticity to recover swallowing function: (i) those aimed at stimulating the peripheral oropharyngeal sensory system by chemical, physical or electrical stimulus; and (ii) those aimed at directly stimulating the pharyngeal motor cortex, such as repetitive transcranial magnetic stimulation (rTMS). The study of Park et al. in this issue of Neurogastroenterology and Motility evaluated the effect of rTMS in dysphagic stroke patients and showed a marked improvement in swallow physiology. Other studies also using rTMS showed plastic changes in pharyngeal motor cortical areas relevant to swallowing function. If further randomized controlled trials confirm these initial results, the neurorehabilitation strategies will be introduced to clinical practice sooner rather than later, improving the recovery of dysphagic stroke patients. Progress at last.

There is still no agreement over optimal pharmacological treatment for irritable bowel syndrome (IBS). Patients with IBS and diarrhea (IBS-D) demonstrate both visceral hypersensitivity and impaired colonic motility with increased frequency and amplitude of giant migrating contractions (GMCs) which cause mass movements, propulsion of stools and initiation of defecation. Both antispasmodics and 5-HT3 receptor antagonists can improve the symptoms and the impaired colonic motility of patients with IBS-D though through very different mechanisms. Antispasmodics act by directly relaxing the colonic smooth muscle cells or antagonizing the excitatory neuromuscular neurotransmission. In contrast, the mechanism of action of 5HT3 antagonists is much more complex and subtle as they inhibit the ascending excitatory component of the peristaltic reflex and GMCs. There are some concerns about the safety of 5HT3 antagonists in long-term treatment. Most of the studies on the treatment of IBS have followed the pharmacological strategy of looking for big clinical effects acting on a single receptor/target. We propose a pharmacologic strategy which uses different drugs for pain and dysmotility in the same patient and includes specific drugs acting on smooth muscle cells, neuromuscular transmission, synaptic transmission and intrinsic afferents. The clinical effect on IBS symptoms would be found in the sum of all these smaller effects on multiple targets.

Background The neurotransmitters mediating inhibitory pathways to internal anal sphincter (IAS) have not been fully characterized. Our aim was to assess the putative release of nitric oxide, purines and vasoactive intestinal peptide (VIP) from inhibitory motor neurons (MNs) and their role in the myogenic tone, resting membrane potential (RMP) of smooth muscle cells (SMC), spontaneous inhibitory junction potentials (sIJP), mechanical relaxation, and IJP induced by electrical field stimulation (EFS) or nicotine. Methods Rat IAS strips were studied using organ baths, microelectrodes, and immunohistochemistry. Key Results Internal anal sphincter strips developed active myogenic tone (0.31 g), enhanced and stabilized by prostaglandin F2α (PGF2α). l-NNA (1 mmol L−1) depolarized SMC and increased tone but did not modify sIJP. In contrast, the specific P2Y1 receptor antagonist MRS2500 (1 μmol L−1) did not modify the RMP or the basal tone but abolished sIJP. Electrical field stimulation and nicotine (10 μmol L−1) caused IAS relaxation (−45.9%VS−52.2%), partially antagonized by l-NNA (35%–45%,P ≤ 0.05) and fully abolished by MRS2500 (P ≤ 0.001). Electrical field stimulation induced a biphasic inhibitory junction potential (IJP), the initial fast component was selectively blocked by MRS2500 and the sustained slow component was blocked by l-NNA. Vasoactive intestinal peptide 6–28 (0.1 μmol L−1) or α-chymotrypsin (10 U mL−1) did not modify the RMP, sIJP, EFS-induced IJP, or relaxation. P2Y1 receptors were immunolocalized in the circular SMC of IAS. Conclusions & Inferences The effects of inhibitory MNs on rat IAS are mediated by a functional co-transmission process involving nitrergic and purinergic pathways through P2Y1 receptors with specific and complementary roles on the control of tone, sIJP, and hyperpolarization and relaxation of IAS following stimulation of inhibitory MNs.

Hydrogen sulphide (H2S) has been recently proposed as a transmitter in the brain and peripheral tissues. Its role in the gastrointestinal tract is still unknown despite some data which suggest an involvement mediating smooth muscle relaxation. The aim of this study was to investigate the effect of this gas on intestinal segments from mouse jejunum and colon, and muscular strips from the human and rat colon. In isolated segments of mouse colon and jejunum, bath applied sodium hydrogen sulphide (NaHS) (a H2S donor) caused a concentration-dependent inhibition of spontaneous motor complexes (MCs) (IC(50) 121 micromol L(-1) in the colon and 150 micromol L(-1) in the jejunum). This inhibitory effect of NaHS on MCs was (i) unaffected by tetrodotoxin (TTX), capsaicin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and N-nitro-L-arginine suggesting a non-neural effect and (ii) significantly reduced by apamin 3 micromol L(-1). NaHS concentration-dependently inhibited the spontaneous motility in strips from human colon (IC(50) 261 micromol L(-1)) and rat colon (IC(50) 31 micromol L(-1)). The inhibitory effect of NaHS on colonic strips was (i) unaffected by the neural blocker TTX (1 micromol L(-1)) with IC(50) 183 micromol L(-1) for the human colon and of 26 micromol L(-1) for the rat colon and (ii) significantly reduced by glybenclamide (10 micromol L(-1)), apamin (3 micromol L(-1)) and TEA (10 mmol L(-1)) with IC(50) values of 2464, 1307 and 2421 micromol L(-1) for human strips, and 80, 167 and 674 micromol L(-1) for rat strips respectively. We conclude that H2S strongly inhibits in vitro intestinal and colonic motor patterns. This effect appears to be critically dependent on K channels particularly apamin-sensitive SK channels and glybenclamide-sensitive K (ATP) channels.

Previous studies have shown that the proximal striated muscle oesophagus is less compliant and more sensitive than the distal smooth muscle oesophagus. Conventional and high resolution manometry described a transition zone between striated and smooth muscle oesophagus. We aimed to evaluate oesophageal tone and sensitivity at the transition zone of oesophagus in healthy volunteers. In 18 subjects (seven men, mean age: 28 years) an oesophageal barostat study was performed. Tone and sensitivity were assessed using stepwise isobaric distensions with the balloon located at transition zone and at distal oesophagus in random order. To study the effect induced on transition zone by a previous distension at the distal oesophagus and vice versa, identical protocol was repeated after 7 days with inverted order. Initial distension of a region is referred to as 'naif' distension and distension of a region following the distension of the other segment as 'primed' distension. Assessment of three oesophageal symptoms (chest pain, heartburn and 'other') was obtained at the end of every distension step. Compliance was significantly higher in the transition zone than in the distal oesophagus (1.47 +/- 0.14 vs 1.09 +/- 0.09 mL mmHg(-1), P = 0.03) after 'naif' distensions. This difference was not observed during 'primed' distensions. Higher sensitivity at transition zone level was found in 11/18 (61%) subjects compared to 6/18 (33%, P < 0.05) at smooth muscle oesophagus. Chest pain and 'other' symptom were more often induced by distention of the transition zone, whereas heartburn was equally triggered by distension of either region. The transition zone is more complaint and more sensitive than smooth muscle oesophagus.

The distribution of interstitial cells of Cajal(ICC) and neurotransmission were investigated inlower oesophageal sphincter (LES) circular musclestrips from Sprague–Dawley (SD) rats, Ws/Ws mutantrats and their wild-type (+/+) siblings. Intramuscularc-Kit-positive cells, confirmed to be ICC-IM by elec-tron microscopy, were observed throughout bothmuscle layers from SD and +/+ rats. In contrast, c-Kit-positive, ultrastructurally typical ICC-IM were absentin Ws/Ws. LES strips from Ws/Ws rats showed in-creased spontaneous contractile activity. Strips fromSD and +/+ rats, responded to electrical neuronal sti-mulation with a relaxation that was in part L-NNAand in part apamin sensitive, followed by a contrac-tion which was decreased by atropine. In Ws/Ws rats,similar to +/+ rats, neurally mediated relaxation wasL-NNA and apamin sensitive and the contraction wasdecreased by atropine. We conclude that in the ratLES, relaxation is mediated by NO and an apamin-sensitive mediator, and contraction primarily byacetylcholine. Despite the absence of c-Kit-positiveICC, nerve–muscle interaction can be accomplishedlikely by diffusion of neurotransmitters to the smoothmuscle cells. The lack of c-Kit-positive ICC is relatedto an increase in the basal tone and spontaneouscontractile activity. The presence of fibroblast-likeICC in Ws/Ws rats might represent immature ICCwhose possible functions need further investigation.Keywords apamin, c-Kit, interstitial cells of Cajal,lower oesophageal sphincter, nitric oxide, Ws/Ws rat.

Background Oropharyngeal dysphagia (OD) is a highly prevalent symptom in older people. Appropriate oropharyngeal sensory feedback is essential for safe and efficient swallowing. However, pharyngeal sensitivity decreases with advancing age and could play a fundamental role in the physiopathology of swallowing dysfunction associated with aging. We aimed to characterize pharyngeal sensitivity and cortical response to a pharyngeal electrical stimulus in healthy volunteers (HV) and older patients with and without OD. Methods Eight young HV, eight older HV without OD, and 14 older patients with OD were studied by electroencephalography through 32 scalp electrodes. Pharyngeal event-related potentials (ERP) were assessed following electrical stimulation of the pharynx. Sensory and tolerance thresholds to the electrical stimulus and latency, amplitude, and scalp current density of each ERP component were analyzed and compared. An ERP source localization study was also performed using the sLORETA software. Key Results Older participants (with and without OD) presented an increased sensory threshold to pharyngeal electrical stimulation (10.2 ± 1.7 mA and 11.5 ± 1.9 mA respectively), compared with young HV (6.0 ± 1.2 mA). The cortical activation of older HV in response to pharyngeal electrical stimulus was reduced compared with young HV (N2 amplitude: 0.22 ± 0.79 vs −3.10 ± 0.59, P

Background The mechanisms involved in anorexia in frail elderly people remain unclear. The objective of this study was to establish whether fasting and postprandial levels of gastrointestinal peptides, gastrointestinal motility, and hunger are modified by age and frailty. Methods Three groups of subjects were studied: (a) frail elderly (>70 years) persons, (b) non-frail elderly (>70 years) persons, and (c) healthy adults (aged 25–65 years). After an overnight fast, participants ingested a 400 Kcal liquid meal and appetite, hormonal, and gastrointestinal responses were monitored during early (0–60 min) and late (60–240 min) postprandial periods. Key Results Frail persons showed poor nutritional status, sarcopenia, and almost absence of hunger during fasting and postprandial periods. Older persons presented higher levels of glucose and insulin during fasting, enhanced postprandial CCK release in early postprandial period and postprandial hyperglycemia and hyperinsulinemia, but similar ghrelin levels than younger adults. Ultrasound scan showed that the fasting antral area was higher and antral compliance lower in old persons. The paracetamol absorption test showed enhanced postprandial gastric emptying in the frail. Non-gallbladder contractors showed no CCK peak in younger and non-frail groups, but the same high CCK peak as contractors in the frail. Conclusions & Inferences Frailty was associated with anorexia, risk of malnutrition, and sarcopenia. Frail persons showed impaired gastric motility (larger antral area at rest, impaired antral compliance, and enhanced postprandial emptying), impaired gallbladder motility, and fasting and/or postprandial alterations in CCK, glucose, and insulin release. Further studies are needed to determine if these factors may contribute to anorexia of aging in frail persons.

Background The origin and modulation mechanisms controlling timing and amplitude of esophageal body peristalsis are not fully understood. We aimed to characterize the neurotransmitters involved in the origin and modulation of circular smooth muscle esophageal body (EB) contractions. Methods Responses of porcine EB strips to electrical stimulation of motor neurons (MNs) were assessed in organ baths and with microelectrodes. The effect of antagonists of inhibitory (L-NAME 1 mmol L−1, MRS2179 10 μmol L−1) and excitatory neurotransmitters (atropine 1 μmol L−1; SR140333 1 μmol L−1-NK1ra-, GR94800 1 μmol L−1-NK2ra-) and of ganglionic neurotransmitters (hexamethonium 100 μmol L−1, ondansetron 1 μmol L−1, NF279 10 μmol L−1) were characterized. Key Results Electrical field stimulation (EFS) induced a frequency-dependent off-contraction (16.8 ± 0.8 g) following a latency period. Latency was significantly reduced by L-NAME (−66.1 ± 4.1%) and MRS2179 (−25.9 ± 5.6%), and strongly increased by atropine (+36.8 ± 5.8%). Amplitude was reduced by L-NAME (−69.9 ± 10.4%), MRS2179 (−34.1 ± 6.0%), atropine (−42.3 ± 4.7%), hexamethonium (−18.9 ± 3.3%), NF279 (−20.7 ± 3.5%), ondansetron (−16.3 ± 3.2%), GR94800 (−28.0 ± 4.8%) SR140333 (−20.9 ± 7.1%), and α-chymotrypsin (−31.3 ± 7.0%). The EFS induced a monophasic nitrergic inhibitory junction potential. Conclusions & Inferences Our results suggest that timing (latency) and amplitude of esophageal contractions are determined by a balance of complex interactions between excitatory and inhibitory MNs. Latency depends on the activation of inhibitory MNs releasing NO and a minor purinergic contribution through P2Y1 receptors, and excitatory MNs releasing ACh. Amplitude depends on a major contribution of excitatory MNs releasing ACh and tachykinins, and also on inhibitory MNs releasing NO, ATP or related purines, and peptidergic neurotransmitters acting as strong modulators of the excitatory neuroeffector transmission.

Background In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y1 receptor. The P2Y12 antagonist AR-C66096 was tested as well. Using this pharmacological approach, we tested whether β-nicotinamide adenine dinucleotide (β-NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon. Methods We carried out muscle bath and microelectrode experiments on circular strips from the human colon and calcium imaging recordings on HEK293 cells, which constitutively express the human P2Y1 receptor. Key Results Both the fast component of IJP and non-nitrergic relaxation was concentration-dependently inhibited by MRS2279 and MRS2500. This antagonism was confirmed in HEK293 cells. However, AR-C66096 did not modify either inhibitory response. Adenosine 5′-Ο-2-thiodiphosphate and MRS2365 caused a smooth muscle hyperpolarization and transient inhibition of spontaneous motility that was antagonized by MRS2279 and MRS2500. β-Nicotinamide adenine dinucleotide inhibited the spontaneous motility (IC50 = 3.3 mmol L−1). Nevertheless, this effect was not antagonized by high concentrations of P2Y1 antagonists. Conclusions & Inferences Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y1 receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y1 antagonists is MRS2500 > MRS2279 > MRS2179. We found that β-NAD partially fulfills the criteria to be considered an inhibitory neurotransmitter in the human colon, but the relative contribution of each purine (ATP/ADP vsβ-NAD) requires further studies.

Background Oropharyngeal dysphagia is a major complaint among the elderly. Our aim was to assess the pathophysiology of oropharyngeal dysphagia in frail elderly patients (FEP). Methods A total of 45 FEP (81.5 ± 1.1 years) with oropharyngeal dysphagia and 12 healthy volunteers (HV, 40 ± 2.4 years) were studied using videofluoroscopy. Each subject’s clinical records, signs of safety and efficacy of swallow, timing of swallow response, hyoid motion and tongue bolus propulsion forces were assessed. Key Results Healthy volunteers presented a safe and efficacious swallow, faster laryngeal closure (0.157 ± 0.013 s) upper esophageal sphincter opening (0.200 ± 0.011 s), and maximal vertical hyoid motion (0.310 ± 0.048 s), and stronger tongue propulsion forces (22.16 ± 2.54 mN) than FEP. By contrast, 63.63% of FEP presented oropharyngeal residue, 57.10%, laryngeal penetration and 17.14%, tracheobronchial aspiration. Frail elderly patients with impaired swallow safety showed delayed laryngeal vestibule (LV) closure (0.476 ± 0.047 s), similar bolus propulsion forces, poor functional capacity and higher 1-year mortality rates (51.7%vs 13.3%, P = 0.021) than FEP with safe swallow. Frail elderly patients with oropharyngeal residue showed impaired tongue propulsion (9.00 ± 0.10 mN), delayed maximal vertical hyoid motion (0.612 ± 0.071 s) and higher (56.0%vs 15.8%, P = 0.012) 1-year mortality rates than those with efficient swallow. Conclusion & Inferences Frail elderly patients with oropharyngeal dysphagia presented poor outcome and high mortality rates. Impaired safety of deglutition and aspirations are mainly caused by delayed LV closure. Impaired efficacy and residue are mainly related to weak tongue bolus propulsion forces and slow hyoid motion. Treatment of dysphagia in FEP should be targeted to improve these critical events.

The mechanisms of stimulation of inhibitory and excitatory motor neurons (MNs) in the lower oesophageal sphincter (LOS) are not fully understood. The aim of this study was to assess the effect of selective stimulation of inhibitory and excitatory MNs in porcine LOS through nicotinic acetylcholine receptors (nAChRs), 5-HT(3) and P2X receptors. Circular LOS strips from adult pigs were studied in organ baths. We compared the effects of stimulation of MNs by electrical field stimulation (26 V, 0.3-20 Hz); nicotine (1-300 micromol L(-1)); 5-HT and 2-Me-5-HT (1 nmol(-1)-30 micromol L(-1)); and alpha,beta-methylene ATP (alpha,beta-meATP 1-100 micromol L(-1)); in standard Krebs solution; a non-adrenergic non-nitrergic non-purinergic (NANNNP) solution; and a non-adrenergic non-cholinergic (NANC) solution. Electrical stimulation of inhibitory MNs caused an intense LOS relaxation (-78.94 +/- 4.50% of LOS tone); and of excitatory MNs, a strong contraction (17.89 +/- 1.96 g). Nicotine 100 micromol L(-1) relaxed LOS (-84.67 +/- 3.98%) in standard Krebs solution, an effect reduced by Tetrodotoxin (TTX) 1 micromol L(-1). Nicotine induced a weak TTX-sensitive contraction (1.64 +/- 0.4 g) in NANNNP solution. 5-HT 10 micromol L(-1) and 2-Me-5-HT 30 micromol L(-1) contracted LOS in standard, NANC and NANNNP conditions, maximal responses (7.30 +/- 1.52 g, 3.50 +/- 0.18 g respectively) being reduced by TTX. alpha,beta-meATP 100 micromol L(-1) caused a LOS relaxation (-17.45 +/- 6.62%) unaffected by TTX in NANC solution, and a contraction (6.7 +/- 0.85 g) antagonized by TTX in NANNNP solution. Our results suggest selective mechanisms for stimulation of intrinsic excitatory and inhibitory motor pathways in porcine LOS. Inhibitory MNs are strongly stimulated by nAChRs and do not respond to stimulation of 5-HT(3) and P2X receptors. By contrast, excitatory MNs are stimulated through 5-HT(3) and P2X receptors, stimulation through nACRs being difficult and causing a weak response.