Over the past decade, research involving the bioengineering of esophageal tissue replacements for repair of congenital defects, cancer, and caustic injuries has advanced rapidly. This is due to the development of innovative biomaterials combined with stem cells that recapitulate tissue ultrastructure, mechanics, and biochemical properties. However, a limitation in the field is a lack of data demonstrating development of innervated tissue exhibiting peristalsis. Currently, no clinically available stem cell therapies/esophageal tissue substitutes exist that restore motility.

This review will discuss advances and limitations in the assessment of esophageal motility in bioengineered tissues along with metrics of success. Additionally, innovative technologies (i.e., 3D bioprinting, electrospinning, and AI) and neuronal cellular approaches for promoting gut innervation will be highlighted to reveal their use for the development of clinical therapies for esophageal replacement. Future directions for development of patient-specific implants will also be discussed to emphasize the importance of access to all populations.

The signaling mechanisms of nucleotides and nucleosides have been extensively studied over the past decades in various conditions affecting distinct organs and tissues. It is well-established that purinergic receptors are expressed in healthy tissues, with expression levels often increasing under pathological conditions. These receptors play crucial roles in numerous physiological and pathological processes, including inflammation, tissue repair, and cellular signaling. However, the purinergic context in the esophagus and its associated pathologies remains poorly understood, representing a significant gap in current knowledge. In this review, we compiled and analyzed the available data on the involvement of P2 purinergic receptors in esophageal diseases, such as gastroesophageal reflux disease and esophageal carcinoma. Specifically, we discuss the pharmacological modulation, functional characterization, and expression patterns of these receptors in various esophageal cell lines and immune tissue samples, under both healthy and pathological conditions. Understanding the mechanisms of action and signaling pathways involving P2 purinergic receptors in the esophagus can offer valuable insights into their biological roles and emphasize their potential as therapeutic targets for future clinical applications.

Background: The current treatment of gastroesophageal reflux disease (GERD) is focused on decreasing gastric acid secretion. However, there is still a group of patients that do not respond to conventional therapy. Proteinase-activated receptors and purinergic receptors have been implicated in inflammation, visceral hyperalgesia and esophageal hypersensitivity. The aim of this study was to evaluate the esophageal expression of PAR2 (F2RL1) and P2RX2, P2RX3 and P2RY2 in GERD patients. Methods: A total of 53 patients with GERD and 9 healthy controls were enrolled in this study. The expression of the studied receptors was quantified using real-time PCR on esophageal biopsies from the patients with GERD and healthy controls. The correlation between the dilated intracellular spaces (DIS) score and patients' quality of life was investigated. Results: PAR2 receptor expression was higher in ERD compared to NERD and controls (326.10 ± 112.30 vs. 266.90 ± 84.76 vs. 77.60 ± 28.50; NS). P2X2 exhibited the highest expression in NERD compared to ERD and controls (302.20 ± 82.94 vs. 40.18 ± 17.78 vs. 26.81 ± 10.27), similarly to P2Y2, which expression was higher in NERD than in ERD and controls (7321.00 ± 1651.00 vs. 5306.0 ± 1738.00 vs. 3476.00 ± 508.0). Conclusions: We found that the expression of F2RL1, P2RX2 and P2RY2 is positively correlated to the DIS score in GERD patients. Higher PAR2, P2X2 and P2Y2 expression could mediate the sensitization of the esophagus and may be associated with the higher intensity of symptoms perceived by NERD patients.

Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract, yet their function in the esophagus remains unknown. The mouse esophagus has been described as primarily skeletal muscle; however, ICC  have been identified in this region. This study characterizes the distribution of skeletal and smooth muscle cells (SMCs) and their spatial relationship to ICC, PDGFRα+ cells, and intramuscular motor neurons in the mouse esophagus. SMCs occupied approximately 30% of the distal esophagus, but their density declined in more proximal regions. Similarly, ANO1+ intramuscular ICC (ICC-IM) were distributed along the esophagus, with density decreasing proximally. While ICC-IM were closely associated with SMCs, they were also present in regions of skeletal muscle. Intramuscular, submucosal, and myenteric PDGFRα+ cells were densely distributed throughout the esophagus, yet only intramuscular PDGFRα+ cells in the lower esophageal sphincter (LES) and distal esophagus expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with intramuscular nNOS+, VIP+, VAChT+, and TH+ neurons and GFAP+ cells resembling intramuscular enteric glia. These findings suggest that ICC-IM and PDGFRα+ cells may have roles in regulating esophageal motility due to their close proximity to each other and to skeletal muscle and SMCs, although further functional studies are needed to explore their role in this region. The mixed muscular composition and presence of interstitial cells in the mouse distal esophagus is anatomically similar to the transitional zone found in the human esophagus, and therefore, motility studies in the mouse may be translatable to humans.

Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα+ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1+ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα+ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα+ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with nNOS+, VIP+, VAChT+ and TH+ neurons throughout the LES and distal esophagus. GFAP+ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα+ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.

Hydrogen sulfide (H2S) is a significant physiologic inhibitory neurotransmitter. The main goal of this research was to examine the contribution of diverse potassium (K+) channels and nitric oxide (NO) in mediating the H2S effect on electrical field stimulation (EFS)-induced neurogenic contractile responses in the lower esophageal sphincter (LES). EFS-induced contractile responses of rabbit isolated LES strips were recorded using force transducers in organ baths that contain Krebs-Henseleit solutions (20 ml). Cumulative doses of NaHS, L-cysteine, PAG, and AOAA were evaluated in NO-dependent and NO-independent groups. The experiments were conducted again in the presence of K+ channel blockers. In both NO-dependent and NO-independent groups, NaHS, L-cysteine, PAG, and AOAA significantly reduced EFS-induced contractile responses. In the NO-dependent group, the effect of NaHS and L-cysteine decreased in the presence of 4-AP, and also the effect of NaHS decreased in the NO-dependent and independent group in the presence of TEA. In the NO-independent group, K+ channel blockers didn't change L-cysteine-induced relaxations. K+ channel blockers had no impact on the effects of PAG and AOAA. In addition, NaHS significantly relaxed 80-mM KCl-induced contractions, whereas L-cysteine, PAG, and AOAA did not. In the present study, H2S decreased the amplitudes of EFS-induced contraction responses. These results suggest that Kv channels and NO significantly contribute to exogenous H2S and endogenous H2S precursor L-cysteine inhibitory effect on lower esophageal sphincter smooth muscle.

Achalasia, characterised by the absence of peristalsis and failure of relaxation of the lower oesophageal sphincter, is an uncommon degenerative condition that results in dysphagia. If left untreated it can lead to aspiration, oesophageal perforation, oesophagitis and malnutrition. It has a range of immune, allergic, viral and genetic aetiological causes. Successful diagnosis relies on the use of oesophagogastroduodenoscopy, barium swallow and oesophageal manometry to characterise the severity of the disease and to rule out underlying malignancy. Although no treatment can reverse the degenerative process, therapeutic strategies including lifestyle modification, medication, endoscopic and operative intervention can help to reduce symptoms. This article reviews the latest methods used to investigate and manage achalasia.

The lower esophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the esophagus. LES smooth muscle cells (SMCs) display depolarized membrane potentials facilitating activation of L-type Ca²⁺ channels. Interstitial cells of Cajal (ICC) express Ca²⁺ -activated Cl^- channels encoded by Ano1 in mouse and monkey LES. Ca²⁺ signaling in ICC activates ANO1 currents in ICC. ICC displayed spontaneous Ca²⁺ transients in mice from multiple firing sites in each cell and no entrainment of Ca²⁺ firing between sites or between cells. Inhibition of ANO1 channels with a specific antagonist caused hyperpolarization of mouse LES and inhibition of tone in monkey and mouse LES muscles. Our data suggest a novel mechanism for LES tone in which Ca²⁺ transient activation of ANO1 channels in ICC generates depolarizing inward currents that conduct to SMCs to activate L-type Ca²⁺ currents, Ca²⁺ entry and contractile tone.

The lower esophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarized, facilitating activation of Ca_v 1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC-IM), through activation of Ca²⁺ -activated-Cl^- channels (ANO1), set membrane potentials of SMCs favorable for activation of Ca_v 1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC-IM are activated by Ca²⁺ transients, but no studies have examined Ca²⁺ dynamics in ICC-IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC-IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1 are expressed in c-Kit⁺ cells (ICC-IM) in mouse and monkey LES clasp muscles. Ca²⁺ signaling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit-KI-GCaMP6f). ICC-IM exhibited spontaneous Ca²⁺ transients from multiple firing sites. Ca²⁺ transients were abolished by CPA or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca²⁺ transients depended on Ca²⁺ influx and store reloading, as Ca²⁺ transient frequency was reduced in Ca²⁺ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Ca_v 1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca²⁺ activates ANO1 channels in ICC-IM in the LES. Coupling of ICC-IM to SMCs drives depolarization, activation of Ca_v 1.2 channels, Ca²⁺ entry and contractile tone. Abstract figure legend Proposed mechanism for generation of contractile tone in the lower esophageal sphincter (LES). Interstitial cells of Cajal (ICC) in the LES generate spontaneous, stochastic Ca²⁺ transients via Ca²⁺ release from the endoplasmic reticulum (ER). The Ca²⁺ transients activate ANO1 Cl- channels causing Cl- efflux (inward current). ANO1 currents have a depolarizing effect on ICC (+++s inside membrane) and this conducts through gap junctions (GJ) to smooth muscle cells (SMCs). Input from thousands of ICC results in depolarized membrane potentials (-40 to -50 mV) which is within the window current range for L-type Ca²⁺ channels. Activation of these channels causes Ca²⁺ influx, activation of contractile elements (CE) and development of tonic contraction. This article is protected by copyright. All rights reserved.

Tobacco smoking-related diseases are estimated to kill more than 8 million people/year and most smokers are willing to stop smoking. The pharmacological approach to aid smoking cessation comprises nicotine replacement therapy (NRT) and inhibitors of the nicotinic acetylcholine receptor, which is activated by nicotine. Common side effects of oral NRT products include hiccoughs, gastrointestinal disturbances and, most notably, irritation, burning and pain in the mouth and throat, which are the most common reasons for premature discontinuation of NRT and termination of cessation efforts. Attempts to reduce the unwanted sensory side effects are warranted, and research discovering the most optimal masking procedures is urgently needed. This requires a firm mechanistic understanding of the neurobiology behind the activation of sensory nerves and their receptors by nicotine. The sensory nerves in the oral cavity and throat express the so-called transient receptor potential (TRP) channels, which are responsible for mediating the nicotine-evoked irritation, burning and pain sensations. Targeting the TRP channels is one way to modulate the unwanted sensory side effects. A variety of natural (Generally Recognized As Safe [GRAS]) compounds interact with the TRP channels, thus making them interesting candidates as safe additives to oral NRT products. The present narrative review will discuss (1) current evidence on how nicotine contributes to irritation, burning and pain in the oral cavity and throat, and (2) options to modulate these unwanted side-effects with the purpose of increasing adherence to NRT. Nicotine provokes irritation, burning and pain in the oral cavity and throat. Managing these side effects will ensure better compliance to oral NRT products and hence increase the success of smoking cessation. A specific class of sensory receptors (TRP channels) are involved in mediating nicotine's sensory side effects, making them to potential treatment targets. Many natural (Generally Recognized As Safe [GRAS]) compounds are potentially beneficial modulators of TRP channels.

An important feature of the gastrointestinal (GI) muscularis externa is its ability to generate phasic contractile activity. However, in some GI regions, a more sustained contraction, referred to as "tone," also occurs. Sphincters are muscles oriented in an annular manner that raise intraluminal pressure, thereby reducing or blocking the movement of luminal contents from one compartment to another. Spontaneous tone generation is often a feature of these muscles. Four distinct smooth muscle sphincters are present in the GI tract: the lower esophageal sphincter (LES), the pyloric sphincter (PS), the ileocecal sphincter (ICS), and the internal anal sphincter (IAS). This chapter examines how tone generation contributes to the functional behavior of these sphincters. Historically, tone was attributed to contractile activity arising directly from the properties of the smooth muscle cells. However, there is increasing evidence that interstitial cells of Cajal (ICC) play a significant role in tone generation in GI muscles. Indeed, ICC are present in each of the sphincters listed above. In this chapter, we explore various mechanisms that may contribute to tone generation in sphincters including: (1) summation of asynchronous phasic activity, (2) partial tetanus, (3) window current, and (4) myofilament sensitization. Importantly, the first two mechanisms involve tone generation through summation of phasic events. Thus, the historical distinction between "phasic" versus "tonic" smooth muscles in the GI tract requires revision. As described in this chapter, it is clear that the unique functional role of each sphincter in the GI tract is accompanied by a unique combination of contractile mechanisms.

Paroxysmal nocturnal hemoglobinuria (PNH) patients display exaggerated intravascular hemolysis and esophageal disorders. Since excess hemoglobin in the plasma causes reduced nitric oxide (NO) bioavailability and oxidative stress, we hypothesized that esophageal contraction may be impaired by intravascular hemolysis. This study aimed to analyze the alterations of the esophagus contractile mechanisms in a murine model of exaggerated intravascular hemolysis induced by phenylhydrazine (PHZ). For comparative purposes, sickle cell disease (SCD) mice were also studied, a less severe intravascular hemolysis model. Esophagus rings were dissected free and placed in organ baths. Plasma hemoglobin was higher in PHZ compared with SCD mice, as expected. The contractile responses produced by carbachol (CCh), KCl, and electrical-field stimulation (EFS) were superior in PHZ esophagi compared with control but remained unchanged in SCD mice. Preincubation with the NO-independent soluble guanylate cyclase stimulator 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272; 1 μM) completely reversed the increased contractile responses to CCh, KCl, and EFS in PHZ mice, but responses remained unchanged with prior treatment with NO donor sodium nitroprusside (300 μM). Protein expression of 3-nitrotyrosine and 4-hydroxynonenal increased in esophagi from PHZ mice, suggesting a state of oxidative stress. In endothelial nitric oxide synthase gene-deficient mice, the contractile responses elicited by KCl and CCh were increased in the esophagus but remained unchanged with the intravascular hemolysis induced by PHZ. In conclusion, our results show that esophagus hypercontractile state occurs in association with lower NO bioavailability due to exaggerated hemolysis intravascular and oxidative stress. Moreover, our study supports the hypothesis that esophageal disorders in PNH patients are secondary to intravascular hemolysis affecting the NO-cGMP pathway.

Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα⁺ cells that were closely associated with nNOS⁺ /VIP⁺ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα⁺ cells.

Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-chymotrypsin-sensitive component was identified in the IAS consistent with vasoactive intestinal peptide-ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα⁺ /SK3⁺ cells were closely aligned with nNOS⁺ /VIP⁺ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα⁺ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα⁺ cells.

Irritable bowel syndrome (IBS), a common gastrointestinal (GI) disorder, is associated with various factors, including lifestyle, infection, stress, intestinal flora, and related diseases. The pharmacotherapeutic stimulation of receptors and downstream signaling pathways is effective in reducing IBS symptoms; however, it is still associated with adverse effects. Various receptors related to GI motility and visceral hypersensitivity should be considered to enhance the benefit/risk ratio of IBS treatments. This review discusses recent pharmacological advances in IBS management. Several receptors related to GI motility and abdominal pain are investigated in various angles. 5-Hydroxytryptamine (5-HT) is an important neurotransmitter that activates the colonic mucosal 5-HT₄ receptor without causing severe cardiovascular adverse effects. The clinical potential of ramosetron for diarrhea-predominant IBS has been suggested because of a lower risk of ischemic colitis than conventional 5-HT₃ receptor antagonists. Toll-like receptors (TLRs), especially TLR2 and TLR4, show a significant effect on the post-infection symptoms and lipopolysaccharide-mediated regulation of GI motility. Histamine is a well-known nitrogenous compound that regulates inflammatory responses and visceral hypersensitivity. Histamine 1 receptor-mediated sensitization of the transient receptor potential vanilloid 1 is associated with IBS. Pharmacological approaches based on these signaling pathways could be useful in the development of novel IBS treatments.

P2Y1 receptors mediate nerve mediated purinergic inhibitory junction potentials (IJP) and relaxations in the gastrointestinal (GI) tract in a wide range of species including rodents and humans. A new P2Y1 antagonist, with a non-nucleotide structure, BPTU, has recently been described using X-ray crystallography as the first allosteric G-protein-coupled receptor antagonist located entirely outside of the helical bundle. In this study, we tested its effect on purinergic responses in the gastrointestinal tract of rodents using electrophysiological and myographic techniques. BPTU concentration dependently inhibited purinergic inhibitory junction potentials and inhibition of spontaneous motility induced by electrical field stimulation in the colon of rats (EC50 = 0.3 μM) and mice (EC50 = 0.06 μM). Mechanical inhibitory responses were also concentration-dependently blocked in the stomach of both species. Compared to MRS2500, BPTU displays a lower potency. In the rat colon nicotine induced relaxation was also blocked by BPTU. BPTU also blocked the cessation of spontaneous contractility elicited by ADPβS and the P2Y1 agonist MRS2365. We conclude that BPTU is a novel antagonist with different structural and functional properties than nucleotidic antagonists that is able to block the P2Y1 receptor located at the neuromuscular junction of the GI tract.

The high-pressure zone of the gastroesophageal junction acts as a multifunctional valve that comprises different groups of smooth muscles located in the distal esophagus and the proximal stomach, in addition to the extrinsic crural diaphragm, composed of skeletal muscle. In this review article, we evaluate the current literature with respect to human subjects, discussing the anatomic locations and physiologic and pharmacologic processes controlling these muscles. These muscles work individually and as a group to prevent reflux of gastric contents while allowing anterograde passage of food and liquid and retrograde passage of gas. We also reviewed new findings with respect to abnormalities that are permissive of reflux of gastric contents into the esophagus, which may lead to gastroesophageal reflux disease.

Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.

Dysphagia is a symptom of swallowing dysfunction that occurs between the mouth and the stomach. Although oropharyngeal dysphagia is a highly prevalent condition (occurring in up to 50% of elderly people and 50% of patients with neurological conditions) and is associated with aspiration, severe nutritional and respiratory complications and even death, most patients are not diagnosed and do not receive any treatment. By contrast, oesophageal dysphagia is less prevalent and less severe, but with better recognized symptoms caused by diseases affecting the enteric nervous system and/or oesophageal muscular layers. Recognition of the clinical relevance and complications of oesophageal and oropharyngeal dysphagia is growing among health-care professionals in many fields. In addition, the emergence of new methods to screen and assess swallow function at both the oropharynx and oesophagus, and marked advances in understanding the pathophysiology of these conditions, is paving the way for a new era of intensive research and active therapeutic strategies for affected patients. Indeed, a unified field of deglutology is developing, with new professional profiles to cover the needs of all patients with dysphagia in a nonfragmented way.

Nerve-mediated relaxation is necessary for the correct accomplishment of gastrointestinal (GI) motility. In the GI tract, NO and a purine are probably released by the same inhibitory motor neuron as inhibitory co-transmitters. The P2Y1 receptor has been recently identified as the receptor responsible for purinergic smooth muscle hyperpolarization and relaxation in the human gut. This finding has been confirmed in P2Y1 -deficient mice where purinergic neurotransmission is absent and transit time impaired. However, the mechanisms responsible for nerve-mediated relaxation, including the identification of the purinergic neurotransmitter(s) itself, are still debatable. Possibly different mechanisms of nerve-mediated relaxation are present in the GI tract. Functional demonstration of purinergic neuromuscular transmission has not been correlated with structural studies. Labelling of purinergic neurons is still experimental and is not performed in routine pathology studies from human samples, even when possible neuromuscular impairment is suspected. Accordingly, the contribution of purinergic neurotransmission in neuromuscular diseases affecting GI motility is not known. In this review, we have focused on the physiological mechanisms responsible for nerve-mediated purinergic relaxation providing the functional basis for possible future clinical and pharmacological studies on GI motility targeting purine receptors.

Motilin agonists promote human gastric motility and cholinergic activity, but excitatory and inhibitory actions are reported in the esophagus. The effect of 5-HT₄ agonists in esophagus is also unclear. Perhaps the use of drugs with additional actions explains the variation. The aim, therefore, was to examine how motilin and prucalopride, selective motilin and 5-HT₄ receptor agonists, modulate neuromuscular functions in human esophagus and gastric fundus.

Electrical field stimulation (EFS) evoked nerve-mediated contractions of circular and longitudinal muscle from human esophageal body and circular muscle from gastric fundus.

In esophageal circular muscle EFS evoked brief contraction, followed by another contraction on termination of EFS, each prevented by atropine. Nitric oxide synthase inhibition facilitated contraction during EFS and the overall contraction became monophasic. In esophagus longitudinal muscle and gastric fundus, EFS evoked cholinergically mediated, monophasic contractions, attenuated by simultaneous nitrergic activation. Motilin (100-300 nM) reduced esophagus circular muscle contractions during EFS, unaffected by L-NAME or apamin. Motilin 300 nM also reduced EFS-evoked contractions of longitudinal muscle. Similar concentrations of motilin facilitated cholinergic activity in the fundus and increased baseline muscle tension. Prucalopride facilitated EFS-evoked contractions in esophagus (tested at 30 μM) and fundus (0.1-30 μM).

Selective motilin and 5-HT₄ agonists have different, region-dependent abilities to modulate human esophageal and stomach neuromuscular activity, exemplified by weak inhibition (motilin) or excitation (5-HT₄) in esophageal body and excitation for both in stomach. In different patients with motility dysfunctions, motilin and 5-HT₄ agonists may reduce gastro-esophageal reflux in different ways.

Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.

This paper reports on gastrointestinal sensitivity, including on the role of refluxate volume on the perception of reflux symptoms; experimental pain models that mimic mechanisms and symptoms of pain associated with esophageal diseases; the potential role of the acid receptor TRPV1 in the genesis of gastroesophageal reflux disease (GERD) symptoms; and roles for ATP and the purine and pyrimidine receptor subfamilies P1, P2X, and P2Y in the pathogenesis of GERD symptoms.

Although gastroesophageal reflux disease (GERD) is a common disorder in Western countries, with a significant impact on quality of life and healthcare costs, the mechanisms involved in the pathogenesis of symptoms remain to be fully elucidated. GERD symptoms and complications may result from a multifactorial mechanism, in which acid and acid-pepsin are the important noxious factors involved. Prolonged contact of the esophageal mucosa with the refluxed content, probably caused by a defective anti-reflux barrier and luminal clearance mechanisms, would appear to be responsible for macroscopically detectable injury to the esophageal squamous epithelium. Receptors on acid-sensitive nerve endings may play a role in nociception and esophageal sensitivity, as suggested in animal models of chronic acid exposure. Meanwhile, specific cytokine and chemokine profiles would appear to underlie the various esophageal phenotypes of GERD, explaining, in part, the genesis of esophagitis in a subset of patients. Despite these findings, which show a significant production of inflammatory mediators and neurotransmitters in the pathogenesis of GERD, the relationship between the hypersensitivity and esophageal inflammation is not clear. Moreover, the large majority of GERD patients (up to 70%) do not develop esophageal erosions, a variant of the condition called non-erosive reflux disease. This summary aims to explore the inflammatory pathway involved in GERD pathogenesis, to better understand the possible distinction between erosive and non-erosive reflux disease patients and to provide new therapeutic approaches.

The following discussion on the physiology of the esophagus includes commentaries on the function of the muscularis mucosa and submucosa as a mechanical antireflux barrier in the esophagus; the different mechanisms of neurological control in the esophageal striated and smooth muscle; new insights from animal models into the neurotransmitters mediating lower esophageal sphincter (LES) relaxation, peristalsis in the esophageal body (EB), and motility of esophageal smooth muscle; differentiation between in vitro properties of the lower esophageal circular muscle, clasp muscle, and sling fibers; alterations in the relationship between pharyngeal contraction and relaxation of the upper esophageal sphincter (UES) in patients with dysphagia; the mechanical relationships between anterior hyoid movement, the extent of upper esophageal opening, and aspiration; the application of fluoroscopy and manometry with biomechanics to define the stages of UES opening; and nonpharmacological approaches to alter the gastroesophageal junction (GEJ).

Pharmacological studies using selective P2Y(1) antagonists, such as MRS2500, and studies with P2Y(1)(-/-) knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y(1) receptors in the colon. The aim of the present study was to test whether P2Y(1) receptors are involved in purinergic neurotransmission in the antrum and cecum.

Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y(1)(-/-) mice.

In the antrum, no differences in resting membrane potential and slow wave activity were observed between groups. In WT animals, electrical field stimulation elicited a MRS2500-sensitive inhibitory junction potential (IJP). In P2Y(1)(-/-) mice, a nitrergic IJP (N(ω) -nitro-l-arginine-sensitive), but not a purinergic IJP was recorded. This IJP was equivalent to the response obtained in strips from WT animals previously incubated with MRS2500. Similar results were obtained in the cecum: 1- the purinergic IJP (MRS2500-sensitive) recorded in WT animals was absent in P2Y(1)(-/-) mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y(1)(-/-) mice 2- MRS2365 a P2Y(1) agonist caused smooth muscle hyperpolarization in WT, but not in P2Y(1) (-/-) animals, and 3- β-NAD caused smooth muscle hyperpolarization both in WT and P2Y(1)(-/-) animals.

1- P2Y(1) receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y(1)(-/-) mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y(1)(-/-) mouse might help in the identification of purinergic neurotransmitter(s).

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.

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) -NK(1) ra-, GR94800 1 μmol L(-1) -NK(2) ra-) and of ganglionic neurotransmitters (hexamethonium 100 μmol L(-1) , ondansetron 1 μmol L(-1) , NF279 10 μmol L(-1) ) were characterized.

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.

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 P2Y(1) 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.