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.

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.

Enteric nervous system neuropathy: repair and restoration.

PURPOSE OF REVIEW: Disordered neurobiology of the enteric nervous system (ENS) underlies a broad assortment of idiopathic, acquired, and congenital pathophysiologies up and down the digestive tract. Progress in two major areas of regenerative medicine related to enteric neuropathy is summarized: new insight into how everyday damage to the ENS might be corrected by indwelling stem cells and prospects for patient-specific replacement of damaged or diseased intestine with one reproduced from pluripotent stem cells derived from embryos or reprogrammed adult cells. RECENT FINDINGS: Germinal centers with undifferentiated stem cells are in position outside ENS ganglia. Messages, which might be released after damage to the ENS or when neurons are lost, direct migration of stem cells into ENS ganglia where they differentiate into one or the other of the specialized classes of interneurons or motor neurons and become 'wired' into the synaptic circuits as neuronal replacements. Action of serotonin and the 5-hydroxytryptamine (HT)4 receptor subtype is a message that initiates the neuronal replacement and circuit restoration process. A reasonable facsimile of a functional intestine can be derived from pluripotent stem cells. SUMMARY: Emerging knowledge of cell and molecular biology of indwelling stem cells in the gut and strategies for application of pluripotential stem cells in patient-specific organ transplantation reflect an emergent revolution in understanding and treating disordered gut function when the underlying cause is ENS neuropathy.

Enteric nervous system: sensory physiology, diarrhea and constipation.

PURPOSE OF REVIEW: The enteric nervous system integrates secretion and motility into homeostatic patterns of behavior susceptible to disorder. Progress in understanding mechanosensory detection in these processes, disordered enteric nervous system integration in diarrhea and constipation and pharmacotherapy is summarized. RECENT FINDINGS: Most neurons in the enteric nervous system discharge in response to distortion. Drugs acting directly to open chloride conductance channels in the mucosal epithelium are therapeutic options for constipation. SUMMARY: Mechanoreception is required for negative feedback control. At issue is identification of the neurons that fulfil the requirement for mechanoreception. Understanding secretomotor neurons is basic to understanding neurogenic secretory diarrhea and constipation and therapeutic strategies. A strategy for treatment of chronic constipation is development of agents that act directly to open Cl channels, which thereby increases the liquidity of the luminal contents. Lubiprostone, a recently Food and Drug Administration-approved drug, increases intraluminal liquidity by opening Cl channels. The future for the drug is clouded by controversy over whether its action is directly at one or the other of chloride channel type 2 (ClC-2) or cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels or both and whether action reflects involvement of G protein-coupled prostaglandin receptors expressed by mucosal epithelial cells.

Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs.

Transient receptor potential A1 (TRPA1) forms nonselective cation channels implicated in acute inflammatory pain and nociception. The mechanism of ligand activation of TRPA1 may involve either covalent modification of cysteine residues or conventional reversible ligand-receptor interactions. For certain electrophilic prostaglandins, covalent modification has been considered as the main mechanism involved in their stimulatory effect on TRPA1. Because some nonsteroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins, we examined several nonelectrophilic NSAIDs on TRPA1 activation using electrophysiological techniques and intracellular Ca(2+) measurements and found that a selected group of NSAIDs can act as TRPA1 agonists. Extracellularly applied flufenamic, niflumic, and mefenamic acid, as well as flurbiprofen, ketoprofen, diclofenac, and indomethacin, rapidly activated rat TRPA1 expressed in Xenopus oocytes and human TRPA1 endogenously expressed in WI-38 fibroblasts. Similarly, the NSAID ligands activated human TRPA1 inducibly expressed in HEK293 cells, but the responses were absent in uninduced and parental HEK293 cells. The response to fenamate agonists was blocked by TRPA1 antagonists, AP-18, HC-030031, and ruthenium red. At subsaturating concentrations, the fenamate NSAIDs also potentiate the activation of TRPA1 by allyl isothiocyanate, cinnamaldehyde, and cold, demonstrating positive synergistic interactions with other well-characterized TRPA1 activators. Importantly, among several thermosensitive TRP channels, the stimulatory effect is specific to TRPA1 because flufenamic acid inhibited TRPV1, TRPV3, and TRPM8. We conclude that fenamate NSAIDs are a novel class of potent and reversible direct agonists of TRPA1. This selective group of TRPA1-stimulating NSAIDs should provide a structural basis for developing novel ligands that noncovalently interact with TRPA1 channels.

Glucagon-like peptide-2 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro.

Glucagon-like peptide-2 (GLP-2) is an important neuroendocrine peptide in intestinal physiology. It influences digestion, absorption, epithelial growth, motility, and blood flow. We studied involvement of GLP-2 in intestinal mucosal secretory behavior. Submucosal-mucosal preparations from guinea pig ileum were mounted in Ussing chambers for measurement of short-circuit current (I(sc)) as a surrogate for chloride secretion. GLP-2 action on neuronal release of acetylcholine was determined with ELISA. Enteric neuronal expression of the GLP-2 receptor (GLP-2R) was studied with immunohistochemical methods. Application of GLP-2 (0.1-100 nM) to the serosal or mucosal side of the preparations evoked no change in baseline I(sc) and did not alter transepithelial ionic conductance. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in I(sc), with an initially rapid rising phase followed by a sustained phase. Application of GLP-2 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-2R antagonist GLP-2-(3-33) significantly reversed suppression of the EFS-evoked responses by GLP-2. Tetrodotoxin, scopolamine, and hexamethonium, but not vasoactive intestinal peptide type 1 receptor (VPAC1) antagonist abolished or reduced to near zero the EFS-evoked responses. GLP-2 suppressed EFS-evoked acetylcholine release as measured by ELISA. Pretreatment with GLP-2-(3-33) offset this action of GLP-2. In the submucosal plexus, GLP-2R immunoreactivity (-IR) was expressed in choline acetyltransferase-IR neurons, somatostatin-IR neurons, neuropeptide Y-IR neurons, and vasoactive intestinal peptide-IR neurons. We conclude that submucosal neurons in the guinea pig ileum express GLP-2R. Activation of GLP-2R decreases neuronally evoked epithelial chloride secretion by suppressing acetylcholine release from secretomotor neurons.

Purinergic signalling in autonomic control.

Intercellular purinergic signalling, which utilizes ATP as a transmitter, is fundamental for the operation of the autonomic nervous system. ATP is released together with 'classical' transmitters from sympathetic and parasympathetic nerves supplying various peripheral targets, modulates neurotransmission in autonomic ganglia, has an important role in local enteric neural control and coordination of intestinal secretion and motility, and acts as a common mediator for several distinct sensory modalities. Recently, the role of ATP-mediated signalling in the central nervous control of autonomic function has been addressed. Emerging data demonstrate that in the brain ATP is involved in the operation of several key cardiorespiratory reflexes, contributes to central processing of viscerosensory information, mediates central CO(2) chemosensory transduction and triggers adaptive changes in breathing, and modulates the activities of the brainstem vagal preganglionic, presympathetic and respiratory neural networks.

Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle.

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.

Effects of bacteria on the enteric nervous system: implications for the irritable bowel syndrome.

A unified scenario emerges when it is considered that a major impact of stress on the intestinal tract is reflected by symptoms reminiscent of the diarrhea-predominant form of irritable bowel syndrome. Cramping abdominal pain, fecal urgency, and explosive watery diarrhea are hallmarks not only of diarrhea-predominant irritable bowel syndrome, but also of infectious enteritis, radiation-induced enteritis, and food allergy. The scenario starts with stress-induced compromise of the intestinal mucosal barrier and continues with microorganisms or other sensitizing agents crossing the barrier and being intercepted by enteric mast cells. Mast cells signal the presence of the agent to the enteric nervous system (ie, the brain-in-the-gut), which uses one of the specialized programs from its library of programs to remove the "threat." This is accomplished by stimulating mucosal secretion, which flushes the threatening agent into the lumen and maintains it in suspension. The secretory response then becomes linked to powerful propulsive motility, which propels the secretions together with the offending agent rapidly in the anal direction. Cramping abdominal pain accompanies the strong propulsive contractions. Urgency is experienced when arrival of the large bolus of liquid distends the recto-sigmoid region and reflexly opens the internal anal sphincter, with continence protection now provided only by central reflexes that contract the puborectalis and external anal sphincter muscles. Sensory information arriving in the brain from receptors in the rapidly distending recto-sigmoid accounts for the conscious sensation of urgency and might exacerbate the individual's emotional stress. The symptom of explosive watery diarrhea becomes self-explanatory in this scenario.

Inhibitory neuromuscular transmission mediated by the P2Y1 purinergic receptor in guinea pig small intestine.

ATP is a putative inhibitory neurotransmitter responsible for inhibitory junction potentials (IJPs) at neuromuscular junctions (IJPs) in the intestine. This study tested the hypothesis that the purinergic P2Y(1) receptor subtype mediates the IJPs. IJPs were evoked by focal electrical stimulation in the myenteric plexus and recorded with "sharp" intracellular microelectrodes in the circular muscle coat. Stimulation evoked three categories of IJPs: 1) purely purinergic IJPs, 2) partially purinergic IJPs, and 3) nonpurinergic IJPs. Purely purinergic IJPs were suppressed by the selective P2Y(1) purinergic receptor antagonist MRS2179. Purely purinergic IJPs comprised 26% of the IJPs. Partially purinergic IJPs (72% of the IJPs) consisted of a component that was abolished by MRS2179 and a second unaffected component. The MRS2179-insensitive component was suppressed or abolished by inhibition of formation of nitric oxide by N(omega)-nitro-l-arginine methyl ester (l-NAME) in some, but not all, IJPs. An unidentified neurotransmitter, different from nitric oxide, mediated the second component in these cases. Nonpurinergic IJPs were a small third category (4%) of IJPs that were abolished by l-NAME and unaffected by MRS2179. Exogenous application of ATP evoked IJP-like hyperpolarizing responses, which were blocked by MRS2179. Application of apamin, which suppresses opening of small-conductance Ca(2+)-operated K(+) channels in the muscle, decreased the amplitude of the purinergic IJPs and the amplitude of IJP-like responses to ATP. The results support ATP as a neurotransmitter for IJPs in the intestine and are consistent with the hypothesis that the P2Y(1) purinergic receptor subtype mediates the action of ATP.

Stimulation of adenosine A1 and A2A receptors by AMP in the submucosal plexus of guinea pig small intestine.

Actions of adenosine 5'-monophosphate (AMP) on electrical and synaptic behavior of submucosal neurons in guinea pig small intestine were studied with "sharp" intracellular microelectrodes. Application of AMP (0.3-100 microM) evoked slowly activating depolarizing responses associated with increased excitability in 80.5% of the neurons. The responses were concentration dependent with an EC(50) of 3.5 +/- 0.5 microM. They were abolished by the adenosine A(2A) receptor antagonist ZM-241385 but not by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid, trinitrophenyl-ATP, 8-cyclopentyl-1,3-dimethylxanthine, suramin, or MRS-12201220. The AMP-evoked responses were insensitive to AACOCF3 or ryanodine. They were reduced significantly by 1) U-73122, which is a phospholipase C inhibitor; 2) cyclopiazonic acid, which blocks the Ca(2+) pump in intraneuronal membranes; and 3) 2-aminoethoxy-diphenylborane, which is an inositol (1,4,5)-trisphosphate receptor antagonist. Inhibitors of PKC or calmodulin-dependent protein kinase also suppressed the AMP-evoked excitatory responses. Exposure to AMP suppressed fast nicotinic ionotropic postsynaptic potentials, slow metabotropic excitatory postsynaptic potentials, and slow noradrenergic inhibitory postsynaptic potentials in the submucosal plexus. Inhibition of each form of synaptic transmission reflected action at presynaptic inhibitory adenosine A(1) receptors. Slow excitatory postsynaptic potentials, which were mediated by the release of ATP and stimulation of P2Y(1) purinergic receptors in the submucosal plexus, were not suppressed by AMP. The results suggest an excitatory action of AMP at adenosine A(2A) receptors on neuronal cell bodies and presynaptic inhibitory actions mediated by adenosine A(1) receptors for most forms of neurotransmission in the submucosal plexus, with the exception of slow excitatory purinergic transmission mediated by the P2Y(1) receptor subtype.

The enteric purinergic P2Y1 receptor.

Significant recent discoveries have shown that the P2Y(1) purinergic receptor subtype is expressed in the enteric nervous system and at intestinal neuromuscular junctions. Secretomotor neurons, which release vasoactive intestinal peptide at their junctions with intestinal secretory glands, express the P2Y(1) receptor. Synaptically released ATP acts at these P2Y(1) receptors to stimulate glandular secretion of electrolytes and H(2)O. Motor neurons in the enteric nervous system release ATP as an inhibitory neurotransmitter at neuromuscular junctions in the intestinal circular muscle coat; this action of ATP is mediated by the P2Y(1) receptor. The emerging evidence for significant involvement of P2Y(1) receptors in local enteric neural control and coordination of intestinal secretion and motility suggests that either the receptors themselves or steps in the post-P2Y(1) receptor signal transduction cascade might be potential therapeutic targets.

Fundamentals of neurogastroenterology: basic science.

The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.

Neurogenic secretion mediated by the purinergic P2Y1 receptor in guinea-pig small intestine.

We tested the hypothesis that ATP is an enteric neurotransmitter that acts at P2Y1 excitatory purinergic receptors on intestinal secretomotor neurons to evoke neurogenic mucosal secretion in the guinea pig. Ussing chamber methods for studying neurogenic intestinal secretion were used to test the hypothesis. Application of ATP evoked concentration-dependent increases in short circuit current (Isc) indicative of stimulation of electrolyte secretion. MRS2179, a selective P2Y1 purinergic receptor antagonist, suppressed the ATP-evoked responses in a concentration-dependent manner with an IC50 of 0.9+/-0.1 microM. Tetrodotoxin or a selective vasoactive intestinal peptide (VPAC1) receptor antagonist suppressed or abolished the ATP-evoked responses. A selective VPAC1 receptor antagonist also suppressed Isc responses evoked by electrical field stimulation of the secretomotor neurons. Secretory responses to ATP were not suppressed by scopolamine, piroxicam nor selective adenosine receptor antagonists. Region-specific differences in responses to ATP corresponded to regional differences in the expression of mRNA transcripts for the P2Y1 receptor. Post-receptor signal transduction for the P2Y1-evoked responses involved stimulation of phospholipase C and an IP3/Ca2+-calmodulin/protein kinase C signaling cascade. Our evidence suggests that ATP is released as a neurotransmitter to stimulate neurogenic mucosal secretion by binding to P2Y1 receptors expressed by VIP-ergic secretomotor neurons.

Distribution and chemical coding of corticotropin-releasing factor-immunoreactive neurons in the guinea pig enteric nervous system.

Immunofluorescence was used to study immunoreactivity (IR) for corticotropin-releasing factor (CRF) in the guinea pig enteric nervous system. CRF-IR was expressed in both the myenteric and the submucosal plexuses of all regions of the large and small intestine and the myenteric plexus of the stomach. CRF-IR nerve fibers were present in the myenteric and submucosal plexuses, in the circular muscle coat, and surrounding submucosal arterioles. Most of the CRF-IR fibers persisted in the myenteric and submucosal plexuses after 7 days in organotypic culture. CRF-IR was not coexpressed with tyrosine hydroxylase-IR or calcitonin gene-related peptide-IR fibers. The proportions of CRF-IR cell bodies in the myenteric plexus increased progressively from the stomach (0.6%) to the distal colon (2.8%). Most of the CRF-IR myenteric neurons (95%) had uniaxonal morphology; the remainder had Dogiel type II multipolar morphology. CRF-IR cell bodies in the myenteric plexus of the ileum expressed IR for choline acetyltransferase (56.9%), substance P (55.0%), and nitric oxide synthase (37.9%). CRF-IR never colocalized with IR for calbindin, calretinin, neuropeptide Y, serotonin, or somatostatin in the myenteric plexus. CRF-IR cell bodies were more abundant in the submucosal plexus (29.9-38.0%) than in the myenteric plexus. All CRF-IR neurons in submucosal ganglia expressed vasoactive intestinal peptide-IR and were likely to be secretomotor/vasodilator neurons. CRF-IR neurons did not express IR for the CRF(1) receptor. CRF(1)-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.

Actions of cysteinyl leukotrienes in the enteric nervous system of guinea-pig stomach and small intestine.

Conventional intracellular microelectrodes, neuronal tracer injection techniques and immunohistochemistry were used to study the actions of cysteinyl leukotrienes (CysLTs) on electrical and synaptic behavior of enteric neurons in guinea-pig stomach and small intestine. Bath application of leukotriene C(4), leukotriene D(4) or leukotriene E(4) evoked a slowly activating depolarizing response in most of the myenteric and submucous plexus neurons in the small intestine while no effect was observed in gastric neurons. The depolarization evoked by cysteinyl leukotrienes in intestinal neurons was associated with increased input resistance and enhanced excitability. Suppression of hyperpolarizing after-potentials occurred in AH type neurons. The depolarizing action of cysteinyl leukotrienes was resistant to tetrodotoxin and cyclooxygenase inhibitors. Neither the CysLT(1) receptor antagonists (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY 171883) and alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV 5901), nor the dual CysLT(1)/CysLT(2) receptor antagonist 6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BAY u9773) significantly altered the depolarizing action of the cysteinyl leukotrienes. Neurotransmission was unaffected by the cysteinyl leukotrienes. The results suggested involvement of cysteinyl leukotrienes in enteric immuno-neural communication through excitatory actions on enteric neurons. The receptor mediating these effects was distinct from currently recognized cysteinyl leukotriene receptor subtypes (CysLT(1) and CysLT(2) receptors) and may represent a new receptor subtype.

Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract.

Electrophysiologic recording and indirect immunofluorescence were combined to study localization of the medium-sized neurofilament 145 (NF145) component of the cytoskeleton in morphologically identified neurons in the myenteric and submucosal plexuses of the guinea pig enteric nervous system. Neuronal localization of chemical markers, including calbindin DK28, calretinin, nitric oxide synthase, choline-acetyltransferase, neuropeptide Y, serotonin, neurokinin 1 receptor protein, and somatostatin, was integrated with electrophysiologic and morphologic results for a more complete assessment. NF145 immunoreactivity (-IR) was present in ganglion cells with Dogiel type I morphology in the myenteric plexus of the stomach and small and large intestine. NF145-IR was not found in myenteric ganglion cells with Dogiel type II morphology. NF145-IR was not present in any of the ganglion cells in the submucosal plexus. NF145 was expressed in nerve fibers in both myenteric and submucosal plexuses. The majority of these fibers were identified as sympathetic postganglionic axons based on their disappearance in organotypic culture and on their expression of tyrosine hydroxylase. The myenteric ganglion cells with NF145-IR had electrophysiologic properties of S-type enteric neurons. NF145-IR was found in neurons with vasoactive intestinal peptide, serotonin, nitric oxide synthase, somatostatin, and neurokinin 1 receptor but not with neuropeptide Y or calbindin. The results in general suggest that NF145 is localized to distinct subsets of myenteric motor neurons and interneurons. Absence of NF145 from ganglion cells in the submucosal plexus is an example of differences between myenteric and submucosal components of the enteric nervous system.