Serotonergic Paracrine Targets in the Intestinal Mucosa.

Serotonin functions as a neurotransmitter in the enteric nervous system. Aside from its neurotransmitter role, serotonin also is a paracrine mediatorial signal in the digestive tract. It is a major paracrine signaling molecule in the integrated physiology of several classes of cells in the intestinal mucosa. Paracrine action can be initiation or suppression of activity in populations of cells that make up divergent phenotypic classes. This underlies phenotypic plasticity in single classes and links single classes to other neighboring phenotypic classes, thereby forming a single and higher-order organization in which different categories of function are integrated to work in harmony as a single homeostatic entity at higher levels of physiological organization. Phenotypic classes of cells that are linked by serotonergic paracrine signaling at upper levels of functional organization in the small intestine are (1) enterochromaffin cells; (2) enteric mast cells; (3) spinal sensory afferents; (4) sympathetic postganglionic neurons; (5) enteric neurons.

Multiple rather than specific autoantibodies were identified in irritable bowel syndrome with HuProt™ proteome microarray.

Immune activation and several autoantibodies might be involved in the pathophysiology of irritable bowel syndrome (IBS). We aimed to identify serum biomarkers for IBS by HuProt™ microarray. IBS patients met Rome III criteria were enrolled. Control groups included healthy controls (HCs) and disease controls (DCs). In stage I, we profiled sera from IBS and control groups with HuProt™ microarrays. Based on significant different proteins in stage I, IBS focused microarrays were constructed and validated in a larger cohort in stage II, then decision tree models were generated to establish a combination of biomarkers. In stage III, 4 purified proteins were verified by ELISA. Finally, we analyzed the correlation of autoantibodies with symptoms. In stage I, we identified 47 significant different proteins including 8 autoantibodies of IgG, 2 of IgA between IBS and HCs; 13 autoantibodies of IgG, 13 of IgA between IBS and DCs. In stage II, we found the positive rates of 14 IgG and IgA autoantibodies in IBS were significantly higher than HCs. Five autoantibodies of IgG and 7 IgA were comprehensively involved in differentiating IBS and HCs with the sensitivity and specificity to diagnose IBS as 40%-46.7% and 79.4%-86.3%. The median optical density value of ELAVL4 (IgG) and PIGP (IgA) were significantly higher in IBS than HCs. Parts of autoantibodies above were related to IBS symptoms. We found a combination of autoantibodies to differentiate IBS with HCs, but no specific autoantibodies could serve as serum biomarkers for IBS.

[Unique characteristics of "the second brain" - The enteric nervous system].

Enteric nervous system (ENS) is composed of intestinal submucosal and myenteric plexuses. ENS may independently regulate intestinal digestive and absorptive function, and it is also known as "the second brain" or gut brain. ENS has significant specificity relative to central nervous system (CNS) in properties and functional activities of neurons and neural circuits. ENS is connected with CNS through the feedback pathway (brain-gut-axis) of sympathetic and parasympathetic nerves and peripheral primary sensory afferent nerves to form the bidirectional brain-gut-axis, which may affect emotion, appetite and behavioral states of individuals. Gastrointestinal functional disorder (GIFD) induced by ENS dysfunction may not only cause abnormal gastrointestinal function but also has been implicated in cognitive and mood disorders, such as irritable bowel syndrome (IBS). GIFD would influence deeply the quality of life in patients. Nevertheless, in the worldwide, ENS has so far received much less attention as compared with CNS. The depth of research and scale of investment in ENS studies have been much lower than those in CNS studies. The situation in China is even more evident. From ENS research history, an outstanding problem is to ignore largely the unique properties of ENS and apply mechanically the hypotheses formed in CNS studies to ENS researches. In this review, the structure and function of ENS are briefly introduced, and the importance of extraordinary characteristics of ENS is illustrated by the problems encountered in our studies.

Serotonergic Integration In the Intestinal Mucosa.

Mucosal serotonin (5-HT) is a key paracrine signaling molecule in the integrated physiology of enterochromaffin cells, enteric mast cells, spinal afferent nerves and the enteric nervous system (ENS). Enterochromaffin cells release 5-HT as a paracrine signal to enteric mast cells, spinal afferents and neurons in the ENS. Enteric mast cells release multiple mediators of paracrine signaling, among which are histamine and the serine proteases, chymase and tryptase, as well as serotonin. Some of these mediators diffuse to receptors on afferent nociceptive and mechanosensitive terminals and sensitize the terminals in a manner that may underlie abdominal pain and distension induced pain in the irritable bowel syndrome. Substance P and calcitonin gene-related peptide (CGRP), released by spinal afferent innervation, degranulate enteric mast cells. Substance P and CGRP are significant factors in mucosal inflammation evoked by bacteria in the colonic microbiome. Binding of immunoglobulin antibodies to FcεRI receptors, on enteric mast cells, degranulate the mast cells and release paracrine mediators that overlay integrative microcircuitry in the ENS. An overlay of histamine "calls up" from the ENS library of programed gut behaviors, a defensive program consisting of a sequence of copious mucosal secretions, increased blood flow and powerful orthograde propulsion organized to move threats out of the colonic lumen. Symptoms of acute watery diarrhea, cramping abdominal pain and incontinence are associated with "running" of the defense program. Intestinal behavioral programs stored in the ENS library are described as working like digital "apps".

Sera with anti-enteric neuronal antibodies from patients with irritable bowel syndrome promote apoptosis in myenteric neurons of guinea pigs and human SH-Sy5Y cells.

BACKGROUND: Sera anti-enteric neuronal antibodies (AENA), neuronal inflammation, and degeneration in myenteric plexus in patients with irritable bowel syndrome (IBS) were reported. Effects of sera AENA in patients with IBS are unclear. METHODS: Patients with IBS met Rome III criteria were enrolled. Controls included healthy subjects (HS) and patients with slow transit functional constipation, inflammatory bowel disease, chronic intestinal pseudo-obstruction, and autoimmune diseases. Indirect immunofluorescence was used to detect AENA. Anti-enteric neuronal antibodies intensities were termed as "1" = weak fluorescence (mild positive); "2" = moderate fluorescence (moderate positive); "3" = very high fluorescence (intensive positive). Intensities of ≥1 were defined as positive and ≥2 were defined as obvious positive. Cultured myenteric neurons of small intestine from guinea pigs and human SH-Sy5Y cells were incubated with fetal bovine serum (FBS), HS sera, or IBS sera with or without AENA. Indirect immunofluorescence with anti-PGP9.5/DAPI/anti-active caspase-3 or TUNEL, Western blot, and flow cytometry were used to detect apoptosis. KEY RESULTS: Overall, 293 patients with IBS were enrolled (41.7 ± 11.5 years). AENA-positive and obvious positive rates in IBS were higher than HS (76.8% vs 33%; 43.7% vs 7%; all P < 0.001). Myenteric neurons incubated with AENA moderate or intensive positive IBS sera showed higher rates of anti-active caspase-3 and TUNEL-positive cells than HS or FBS (20% ± 7.3% and 35% ± 13.3% vs 4.3% ± 1.5% and 0.9% ± 0.4%, respectively; 6.2% ± 2.0% and 10.2% ± 4.6% vs 1.3% ± 1.9% and 0.5%±0.5%, respectively; all P < 0.05). Human SH-Sy5Y cells incubated with AENA moderate or intensive positive IBS sera showed increased cleaved caspase-3 and Bax expression and decreased Bcl-2 expression. Flow cytometry showed apoptosis rates of these two groups were higher than that of AENA mild positive, negative, HS, and FBS (7.6%±0.8% and 10.7%±1.3% vs 5.0%±0.8%, 3.8%±0.3%, 3.4%±0.2% and, 2.8%±0.2%, P < 0.05). CONCLUSIONS AND INFERENCES: The AENA obvious positive rate in patients with IBS was higher than HS, and sera with higher levels of AENA promoted neuronal apoptosis. AENA-mediated neuropathy might exist in a subset of patients with IBS.

Enteric Neurobiology: Discoveries and Directions.

Discovery and documentation of noncholinergic-nonadrenergic neurotransmission in the enteric nervous system started a revolution in mechanisms of neural control of the digestive tract that continues into a twenty-first century era of translational gastroenterology, which is now firmly embedded in the term, neurogastroenterology. This chapter, on Enteric Neurobiology: Discoveries and Directions, tracks the step-by-step advances in enteric neuronal electrophysiology and synaptic behavior and progresses to the higher order functions of central pattern generators, hard wired synaptic circuits and libraries of neural programs in the brain-in-the-gut that underlie the several different patterns of motility and secretory behaviors that occur in the specialized, serially-connected compartments extending from the esophagus to the anus.

Changes in Enteric Neurons of Small Intestine in a Rat Model of Irritable Bowel Syndrome with Diarrhea.

BACKGROUND/AIMS: Physical and/or emotional stresses are important factors in the exacerbation of symptoms in irritable bowel syndrome (IBS). Several lines of evidence support that a major impact of stress on the gastrointestinal tract occurs via the enteric nervous system. We aimed to evaluate histological changes in the submucosal plexus (SMP) and myenteric plexus (MP) of the distal ileum in concert with the intestinal motor function in a rat model of IBS with diarrhea. METHODS: The rat model was induced by heterotypic chronic and acute stress (CAS). The intestinal transit was measured by administering powdered carbon by gastric gavage. Double immunohistochemical fluorescence staining with whole-mount preparations of SMP and MP of enteric nervous system was used to assess changes in expression of choline acetyltransferase, vasoactive intestinal peptide, or nitric oxide synthase in relation to the pan neuronal marker, anti-Hu. RESULTS: The intestinal transit ratio increased significantly from control values of 50.8% to 60.6% in the CAS group. The numbers of enteric ganglia and neurons in the SMP were increased in the CAS group. The proportions of choline acetyltransferase- and vasoactive intestinal peptide-immunoreactive neurons in the SMP were increased (82.1 ± 4.3% vs. 76.0 ± 5.0%, P = 0.021; 40.5 ± 5.9% vs 28.9 ± 3.7%, P = 0.001), while nitric oxide synthase-immunoreactive neurons in the MP were decreased compared with controls (23.3 ± 4.5% vs 32.4 ± 4.5%, P = 0.002). CONCLUSIONS: These morphological changes in enteric neurons to CAS might contribute to the dysfunction in motility and secretion in IBS with diarrhea.

ß-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to ß-nicotinamide adenine dinucleotide (ß-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, ß-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. ß-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of ß-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of ß-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for ß-NAD at intestinal neuromuscular junctions. The data suggest that ß-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of ß-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.

Innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine.

Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca²âº by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals.

Dietary glutamate: interactions with the enteric nervous system.

BACKGROUND/AIMS: Digestion of dietary protein elevates intraluminal concentrations of glutamate in the small intestine, some of which gain access to the enteric nervous system (ENS). Glutamate, in the central nervous system (CNS), is an excitatory neurotransmitter. A dogma that glutamatergic neurophysiology in the ENS recapitulates CNS glutamatergic function persists. We reassessed the premise that glutamatergic signaling in the ENS recapitulates its neurotransmitter role in the CNS. METHODS: Pharmacological analysis of actions of receptor agonists and antagonists in concert with immunohistochemical localization of glutamate transporters and receptors was used. Analysis focused on intracellularly-recorded electrical and synaptic behavior of ENS neurons, on stimulation of mucosal secretion by secretomotor neurons in the submucosal plexus and on muscle contractile behavior mediated by musculomotor neurons in the myenteric plexus. RESULTS: Immunoreactivity for glutamate was expressed in ENS neurons. ENS neurons expressed immunoreactivity for the EAAC-1 glutamate transporter. Neither L-glutamate nor glutamatergic receptor agonists had excitatory actions on ENS neurons. Metabotropic glutamatergic receptor agonists did not directly stimulate neurogenic mucosal chloride secretion. Neither L-glutamate nor the metabotropic glutamatergic receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), changed the mean amplitude of spontaneously occurring contractions in circular or longitudinal strips of intestinal wall from either guinea pig or human small intestinal preparations. CONCLUSIONS: Early discoveries, for excitatory glutamatergic neurotransmission in the CNS, inspired enthusiasm that investigation in the ENS would yield discoveries recapitulating the CNS glutamatergic story. We found this not to be the case.

Mast cell expression of the serotonin1A receptor in guinea pig and human intestine.

Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

Taming the irritable bowel.

This narrative review covers the mechanisms of actions of trendy drugs approved for or proposed for calming the irritable bowel. Many drugs that target functional gastrointestinal disorders (FGIDS), which includes IBS, have their actions in the enteric nervous system (i.e., the brain-in-the-gut). The in-depth insight into fundamental neurophysiology, which is essential for understanding how the drugs act to achieve their effects, is covered from a neurogastroenterological view point. Pharmacotherapeutic research in FGIDS, which is now lagging, is focused mainly on symptom control. Major progress will require a change to orientation on the malfunction underlying each of the symptoms that constitute Manning, Rome I and Rome II symptom-based criteria for FIGD diagnoses. A high incidence of autoimmune degenerative neuropathy in the enteric nervous system occurs in IBS and is postulated to be the cause of symptoms emerging from failure of normal neural control of motility, blood flow and secretory glands, in concert with sensitization of spinal and vagal sensory mechanisms.

CaMKII is essential for the function of the enteric nervous system.

BACKGROUND: Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species. METHODOLOGY/PRINCIPAL FINDINGS: CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue. CONCLUSION: ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.

Anti-enteric neuronal antibodies and the irritable bowel syndrome.

BACKGROUND/AIMS: Functional gastrointestinal disorders are those in which no abnormal metabolic or physical processes, which can account for the symptoms, can be identified. The irritable bowel syndrome (IBS) is a significant functional disorder, which affects 10-20 percent of the population worldwide. Predominant symptoms of IBS are abnormal defecation associated with abdominal pain, both of which may be exacerbated by psychogenic stress. Our study was designed to test a hypothesis that symptoms in a subset of patients with a diagnosis of IBS are associated with an autoimmune degenerative neuropathy in the enteric nervous system. METHODS: Serum was collected from Rome II-IBS patients and controls at the University of North Carolina Functional Gastrointestinal Diseases Center. Assay procedures were immunohistochemical localization of antibody binding to enteric neurons and human protein microarray assay for antigens recognized by antibodies in the sera. RESULTS: Eighty-seven percent of IBS sera and 59% of control sera contained anti-enteric neuronal antibodies. Antibody immunostaining was seen in the nucleus and cytoplasm of neurons in the enteric nervous system. Protein microarray analysis detected antibody reactivity for autoantigens in serum with anti-enteric neuronal antibodies and no reactivity for the same autoantigens in samples not containing anti-enteric neuronal antibodies in our immunostaining assay. Antibodies in sera from IBS patients recognized only 3 antigens out of an 8,000 immunoprotein array. The 3 antigens were: (1) a nondescript ribonucleoprotein (RNP-complex); (2) small nuclear ribonuclear polypeptide A; and (3) Ro-5,200 kDa. CONCLUSIONS: Results of the present study suggest that symptoms in a subset of IBS patients might be a reflection of enteric neuronal damage or loss, caused by circulating anti-enteric autoimmune antibodies.

Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro.

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current (I(sc)), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in I(sc) that had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9-39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.

Visceral pain: spinal afferents, enteric mast cells, enteric nervous system and stress.

This review aims to examine current basic and clinical concepts, the results of which are expanding our understanding of visceral hypersensitivity and functional abdominal pain of intestinal origin in relation to the enteric nervous system (ENS), spinal sensory neurons and enteric mast cells. Advances in this sphere are translating to improved insight into chronic functional abdominal and pelvic pain syndromes in general.