ABSTRACT
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.
Subject(s)
Enteric Nervous System , Quality of Life , Brain , China , HumansABSTRACT
Both Docosahexaenoic acid (DHA) and Phosphatidylcholine (PC) have been shown to halt the pathogenesis of Alzheimer disease (AD) and vascular dementia. This study aimed to investigate the role of DHA-containing PC (DHA-PC) in the improvement of Aß25-35-induced cognitive deficits in rats. Aß25-35-induced AD rats were treated for 30 days with DHA-PC, which was extracted from Sthenoteuthis oualaniensis spawns. Cognitive improvement of the AD rats was detected using the Morris water maze (MWM). The results demonstrated that DHA-PC could improve the learning and memory abilities of AD rats in a dose-dependent pattern. Further analyses showed that expression of phosphorylated tau decreased, and the neuronal morphology recovered in brains of DHA-PC-treated AD rats, as compared with mock-treated AD rats. In addition, DHAPC treatment increased the activity of GSH-Px and SOD in the cortex and hippocampus of AD rats. Taken together, these data suggest that DHA-PC is able to improve the cognitive deficits in AD rats, probably through decreasing the phosphorylation of tau in the cortex and hippocampus CA1 area, and increasing the GSH-Px and SOD activities in the brain of AD rats.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Cognition Disorders/drug therapy , Docosahexaenoic Acids/therapeutic use , Peptide Fragments/metabolism , Phosphatidylcholines/therapeutic use , Alzheimer Disease/chemically induced , Alzheimer Disease/complications , Amyloid beta-Peptides/toxicity , Animals , Cognition Disorders/chemically induced , Cognition Disorders/complications , Decapodiformes , Disease Models, Animal , Docosahexaenoic Acids/chemistry , Docosahexaenoic Acids/isolation & purification , Docosahexaenoic Acids/pharmacology , Female , Male , Peptide Fragments/toxicity , Phosphatidylcholines/chemistry , Phosphatidylcholines/isolation & purification , Phosphatidylcholines/pharmacology , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Colon/physiology , Jejunum/physiology , Muscle Contraction , NAD/metabolism , Receptor, Adenosine A1/metabolism , Synaptic Transmission , Adenosine/analogs & derivatives , Adenosine/pharmacokinetics , Adenosine A1 Receptor Agonists/pharmacokinetics , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacokinetics , Animals , Colon/pathology , Deoxyadenine Nucleotides/pharmacokinetics , Electric Stimulation/methods , Guinea Pigs , Humans , Jejunum/pathology , Ligands , Membrane Potentials/physiology , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Smooth/physiology , Neuromuscular Junction/physiology , Purinergic P2Y Receptor Antagonists/pharmacokinetics , Synaptic Transmission/drug effects , Synaptic Transmission/physiologyABSTRACT
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 Ca²âº 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.
Subject(s)
Enteric Nervous System/physiology , Intestine, Small/innervation , Mast Cells/metabolism , Spinal Nerves/physiology , Animals , Cell Degranulation , Chymases/metabolism , Electric Stimulation , Enteric Nervous System/metabolism , Excitatory Postsynaptic Potentials , Guinea Pigs , Histamine Release , Humans , Male , Mast Cells/drug effects , Neurons, Afferent/physiology , Paracrine Communication , Sensory System Agents/pharmacology , Spinal Nerves/metabolism , Substance P/metabolism , Time FactorsABSTRACT
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.
ABSTRACT
The distribution and activity of endomorphins (EMs), which are endogenous µ-opioid receptor (MOR) ligands in the gastrointestinal tract (GI), are yet to be elucidated. The current study aimed to shed light on this topic. EM2 was expressed in the enteric neurons in the myenteric plexus of the mid-colon. Of the EM2-immunoreactive (EM2-IR) neurons, 53 ± 4.6%, 26 ± 4.5%, 26 ± 2.8% and 49 ± 4.2% displayed immunopositive staining for choline acetyl transferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide synthetase (NOS), respectively. A bath application of EM2 (2 µM) enhanced spontaneous contractile amplitude and tension, which were reversed by ß-FNA (an antagonist of MOR) but not NG-nitro-L-arginine methyl ether (L-NAME, a non-selective inhibitor of NOS) or VIP6-28 (an antagonist of the VIP receptor) in the colonic strips. EM2 significantly suppressed inhibitory junction potentials (IJPs) in 14 of the 17 examined circular muscle cells, and this effect was not antagonized by preincubation in L-NAME. EM2 was widely expressed in interneurons and motor neurons in the myenteric plexus and presynaptically inhibited fast IJPs, thereby enhancing spontaneous contraction and tension in the colonic smooth muscle.
ABSTRACT
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.
Subject(s)
Intestinal Mucosa/metabolism , Mast Cells/metabolism , Receptor, Serotonin, 5-HT1A/biosynthesis , Animals , Blotting, Western , Cromolyn Sodium/pharmacology , Enteric Nervous System/metabolism , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Histamine H1 Antagonists/pharmacology , Humans , Immunohistochemistry , Indicators and Reagents , Intestines/cytology , Intestines/drug effects , Ketotifen/pharmacology , Male , Mast Cells/drug effects , Neuroglia/physiology , Neurons/physiology , Phosphodiesterase Inhibitors/pharmacology , Piperazines/pharmacology , Serotonin Antagonists/pharmacology , Tetrodotoxin/pharmacology , Thioxanthenes/pharmacology , Xanthones/pharmacology , p-Methoxy-N-methylphenethylamine/pharmacologyABSTRACT
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.
Subject(s)
Chlorides/metabolism , Glucagon-Like Peptide 1/pharmacology , Intestinal Mucosa/innervation , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Acetylcholine/metabolism , Animals , Carbachol/pharmacology , Choline O-Acetyltransferase/metabolism , Cytoplasm/metabolism , ELAV Proteins/metabolism , Electric Conductivity , Electric Stimulation , Electrophysiological Phenomena/drug effects , Electrophysiological Phenomena/physiology , Glucagon-Like Peptide 1/antagonists & inhibitors , Glucagon-Like Peptide-1 Receptor , Guinea Pigs , Hexamethonium/pharmacology , Ileum/drug effects , Ileum/innervation , Ileum/metabolism , In Vitro Techniques , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Intestine, Small/innervation , Male , Neurons/drug effects , Neurons/metabolism , Neuropeptide Y/metabolism , Peptide Fragments/pharmacology , Receptors, Glucagon/metabolism , Receptors, Vasoactive Intestinal Polypeptide, Type I/antagonists & inhibitors , Scopolamine/pharmacology , Somatostatin/metabolism , Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
BACKGROUND AND AIMS: Treatments with morphine or opioid agonists cause constipation. Lubiprostone is approved for treatment of adult idiopathic constipation and constipation-predominant IBS in adult women. We tested whether lubiprostone can reverse morphine-suppression of mucosal secretion in human intestine and explored the mechanism of action. METHODS: Fresh segments of jejunum discarded during Roux-En-Y gastric bypass surgeries were used. Changes in short-circuit current (ΔIsc) were recorded in Ussing flux chambers as a marker for electrogenic chloride secretion during pharmacological interactions between morphine, prostaglandin receptor antagonists, chloride channel blockers and lubiprostone. RESULTS: Morphine suppressed basal Isc. Lubiprostone reversed morphine suppression of basal Isc. Lubiprostone, applied to the mucosa in concentrations ranging from 3 nM to 30 µM, evoked increases in Isc in concentration-dependent manner when applied to the mucosal side of muscle-stripped preparations. Blockade of enteric nerves did not change stimulation of Isc by lubiprostone. Removal of chloride or application of bumetanide or NPPB suppressed or abolished responses to lubiprostone. Antagonists acting at CFTR channels and prostaglandin EP(4) receptors, but not at E(1), EP(1-3) receptors, partially suppressed stimulation of Isc by lubiprostone. CONCLUSIONS: Antisecretory action of morphine results from suppression of excitability of secretomotor neurons in the enteric nervous system. Lubiprostone, which does not affect enteric neurons directly, bypasses the action of morphine by directly opening mucosal chloride channels.
Subject(s)
Alprostadil/analogs & derivatives , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Morphine/pharmacology , Alprostadil/pharmacology , Analgesics, Opioid/adverse effects , Analgesics, Opioid/pharmacology , Benzoates/pharmacology , Cadmium Chloride/pharmacology , Cathartics/pharmacology , Drug Interactions , Female , Humans , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Lubiprostone , Morphine/adverse effects , Thiazolidines/pharmacology , Tissue Culture TechniquesABSTRACT
Lubiprostone activates ClC-2 chloride channels in epithelia. It is approved for treatment of chronic idiopathic constipation in adults and constipation-predominate irritable bowel syndrome in women. We tested a hypothesis that lubiprostone can reverse the constipating action of morphine and investigated the mechanism of action. Short-circuit current (Isc) was recorded in Ussing chambers as a marker for chloride secretion during pharmacological interactions between morphine and lubiprostone. Measurements of fecal wet weight were used to obtain information on morphine-lubiprostone interactions in conscious mice. Morphine decreased basal Isc, with an IC(50) of 96.1 nM. The action of dimethylphenylpiperazinium (DMPP), a nicotinic receptor agonist that stimulates neurogenic Isc, was suppressed by morphine. Lubiprostone applied after pretreatment with morphine reversed morphine suppression of both basal Isc and DMPP-evoked chloride secretion. Electrical field stimulation (EFS) of submucosal neurons evoked biphasic increases in Isc. Morphine abolished the first phase and marginally suppressed the second phase. Lubiprostone reversed, in concentration-dependent manner, the action of morphine on the first and second phases of the EFS-evoked responses. Subcutaneous lubiprostone increased fecal wet weight and numbers of pellets expelled. Morphine significantly reduced fecal wet weight and number of pellets. Injection of lubiprostone, 30-min after morphine, reversed morphine-induced suppression of fecal wet weight. We conclude that inhibitory action of morphine on chloride secretion reflects suppression of excitability of cholinergic secretomotor neurons in the enteric nervous system. Lubiprostone, which does not directly affect enteric neurons, bypasses the neurogenic constipating effects of morphine by directly opening chloride channels in the mucosal epithelium.
Subject(s)
Alprostadil/analogs & derivatives , Chloride Channels/metabolism , Constipation/prevention & control , Intestinal Mucosa/metabolism , Morphine/adverse effects , Alprostadil/administration & dosage , Alprostadil/pharmacology , Alprostadil/therapeutic use , Animals , CLC-2 Chloride Channels , Chloride Channels/antagonists & inhibitors , Chlorine/metabolism , Constipation/chemically induced , Dose-Response Relationship, Drug , Feces/chemistry , Guinea Pigs , In Vitro Techniques , Intestinal Mucosa/drug effects , Intestines/drug effects , Ion Channel Gating/drug effects , Lubiprostone , Male , Mice , Mice, Inbred C57BL , Morphine/administration & dosage , Neurons/metabolism , Neurons/physiology , Prostaglandins/metabolismABSTRACT
Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.
Subject(s)
Alprostadil/analogs & derivatives , Colon/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Intestine, Small/drug effects , Alprostadil/administration & dosage , Alprostadil/pharmacology , Animals , Biological Transport/drug effects , Cathartics/pharmacology , Chlorides/metabolism , Colon/metabolism , Dose-Response Relationship, Drug , Guinea Pigs , Histamine/metabolism , Intestine, Small/metabolism , Lubiprostone , Neurons/physiology , Prostaglandins/metabolismABSTRACT
The canonical transient receptor potential (TRPC) family of ion channels is implicated in many neuronal processes including calcium homeostasis, membrane excitability, synaptic transmission, and axon guidance. TRPC channels are postulated to be important in the functional neurobiology of the enteric nervous system (ENS); nevertheless, details for expression in the ENS are lacking. Reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry were used to study the expression and localization of TRPC channels. We found mRNA transcripts, protein on Western blots, and immunoreactivity (IR) for TRPC1/3/4/6 expressed in the small intestinal ENS of adult guinea pigs. TRPC1/3/4/6-IR was localized to distinct subpopulations of enteric neurons and was differentially distributed between the myenteric and submucosal divisions of the ENS. TRPC1-IR was widely distributed and localized to neurons with cholinergic, calretinin, and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to both cholinergic and noncholinergic secretomotor neurons in the submucosal plexus. TRPC3-IR was found only in the submucosal plexus and was expressed exclusively by neuropeptide Y-IR neurons. TRPC4/6-IR was expressed in only a small population of myenteric neurons, but was abundantly expressed in the submucosal plexus. TRPC4/6-IR was coexpressed with both cholinergic and nitrergic neurochemical codes in the myenteric plexus. In the submucosal plexus, TRPC4/6-IR was expressed exclusively in noncholinergic secretomotor neurons. No TRPC1/3/4/6-IR was found in calbindin-IR neurons. TRPC3/4/6-IR was widely expressed along varicose nerve fibers and colocalized with synaptophysin-IR at putative neurotransmitter release sites. Our results suggest important roles for TRPC channels in ENS physiology and neuronal regulation of gut function.
Subject(s)
Enteric Nervous System/metabolism , Gastrointestinal Tract/innervation , Neurons/metabolism , TRPC Cation Channels/metabolism , Animals , Blotting, Western , Calcium-Binding Proteins/metabolism , Digestion/physiology , Enteric Nervous System/cytology , Gastrointestinal Tract/physiology , Guinea Pigs , Immunohistochemistry , Male , Myenteric Plexus/cytology , Myenteric Plexus/metabolism , Neurons/cytology , Neuropeptides/metabolism , Neurotransmitter Agents/metabolism , Peristalsis/physiology , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Submucous Plexus/cytology , Submucous Plexus/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Synaptophysin/metabolism , TRPC Cation Channels/geneticsABSTRACT
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.
Subject(s)
Adenosine Triphosphate/metabolism , Ileum/innervation , Jejunum/innervation , Muscle, Smooth/innervation , Myenteric Plexus/metabolism , Neural Inhibition , Neuromuscular Junction/metabolism , Receptors, Purinergic P2/metabolism , Action Potentials , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Anesthetics, Local/pharmacology , Animals , Apamin/pharmacology , Electric Stimulation , Enzyme Inhibitors/pharmacology , Gastrointestinal Motility , Guinea Pigs , Ileum/drug effects , Ileum/metabolism , In Vitro Techniques , Jejunum/drug effects , Jejunum/metabolism , Male , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Neural Inhibition/drug effects , Neuromuscular Junction/drug effects , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Potassium Channel Blockers/pharmacology , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2Y1 , Small-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Tetrodotoxin/pharmacologyABSTRACT
Glutamate is the major fast excitatory transmitter in the central nervous system. While normal synaptic transmission is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, N-methyl-D-aspartate (NMDA) receptors are thought to selectively contribute to plasticity. Genetically enhancing NMDA receptor functions enhances animal behavior in normal physiological learning and enhances their sensitivity in the case of tissue injury. One major mechanism for NMDA receptors is synaptic long-term potentiation (LTP). Here we present evidence that NMDA receptors not only contribute to normal synaptic responses induced by stimulation of local layer V or white matters, but also contribute to generation of action potentials induced by a depolarizing step applied to the soma. Calcium-calmodulin sensitive adenylyl cyclase 1 and cAMP signal pathways likely mediate these effects. Considering the importance of cingulate neurons in nociception and pain, our results provide a new mechanism for NMDA receptor contributing to neuronal synaptic transmission, spiking properties in forebrains, and possible forebrain-related behavioral nociceptive responses and pain.
Subject(s)
Action Potentials , Prosencephalon/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Adenylyl Cyclases/metabolism , Animals , Cyclic AMP/metabolism , MiceABSTRACT
Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.
Subject(s)
Enteric Nervous System/physiology , Intestine, Small/innervation , Intestine, Small/physiology , Platelet Activating Factor/physiology , Animals , Blotting, Western , Calbindins , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophysiology , Enteric Nervous System/metabolism , Excitatory Postsynaptic Potentials/physiology , Female , Gastrointestinal Motility/physiology , Guinea Pigs , Immunohistochemistry , Intestine, Small/metabolism , Male , Membrane Potentials/physiology , Microelectrodes , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/biosynthesis , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium Binding Protein G/biosynthesis , S100 Calcium Binding Protein G/genetics , Sympathetic Nervous System/physiology , Synapses/physiology , Synaptic Transmission/physiologyABSTRACT
Activity-dependent synaptic plasticity is known to be important in learning and memory, persistent pain and drug addiction. Glutamate NMDA receptor activation stimulates several protein kinases, which then trigger biochemical cascades that lead to modifications in synaptic efficacy. Genetic and pharmacological techniques have been used to show a role for Ca2+/calmodulin-dependent kinase II (CaMKII) in synaptic plasticity and memory formation. However, it is not known if increasing CaMKII activity in forebrain areas affects behavioral responses to tissue injury. Using genetic and pharmacological techniques, we were able to temporally and spatially restrict the over expression of CaMKII in forebrain areas. Here we show that genetic overexpression of CaMKII in the mouse forebrain selectively inhibits tissue injury-induced behavioral sensitization, including allodynia and hyperalgesia, while behavioral responses to acute noxious stimuli remain intact. CaMKII overexpression also inhibited synaptic depression induced by a prolonged repetitive stimulation in the ACC, suggesting an important role for CaMKII in the regulation of cingulate neurons. Our results suggest that neuronal CaMKII activity in the forebrain plays a role in persistent pain.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gyrus Cinguli/enzymology , Hyperalgesia/enzymology , Long-Term Synaptic Depression , Prosencephalon/enzymology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Chronic Disease , Electric Stimulation , Gene Expression Regulation, Enzymologic/genetics , Hyperalgesia/genetics , Hyperalgesia/physiopathology , Male , Mice , Mice, Transgenic , Pain Measurement , Pain Threshold/drug effects , Pain, Intractable/enzymology , Pain, Intractable/genetics , Pain, Intractable/physiopathology , Phosphorylation , Physical Stimulation , Synaptic Transmission/genetics , Up-Regulation/geneticsABSTRACT
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.
Subject(s)
Intestine, Small/metabolism , Receptors, Purinergic P2/physiology , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Bumetanide/pharmacology , Chlorides/metabolism , Dose-Response Relationship, Drug , Electric Stimulation , Electrolytes/metabolism , Gene Expression , Guinea Pigs , In Vitro Techniques , Intestine, Small/drug effects , Intestine, Small/innervation , Male , Purinergic P1 Receptor Agonists , Purinergic P1 Receptor Antagonists , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y1 , Receptors, Vasoactive Intestinal Polypeptide, Type I/agonists , Receptors, Vasoactive Intestinal Polypeptide, Type I/antagonists & inhibitors , Signal Transduction/drug effects , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Tetrodotoxin/pharmacology , Theophylline/analogs & derivatives , Theophylline/pharmacology , Triazines/pharmacology , Triazoles/pharmacology , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
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.
Subject(s)
Corticotropin-Releasing Hormone/metabolism , Enteric Nervous System/cytology , Neurons/metabolism , Animals , Calbindin 2 , Calbindins , Calcitonin Gene-Related Peptide/metabolism , Cell Count/methods , Choline O-Acetyltransferase/metabolism , Colchicine/pharmacology , ELAV Proteins/metabolism , Guinea Pigs , Immunohistochemistry/methods , In Vitro Techniques , Male , Myenteric Plexus/cytology , Myenteric Plexus/drug effects , Myenteric Plexus/metabolism , Neurons/chemistry , Neuropeptide Y/metabolism , Nitric Oxide Synthase/metabolism , Phosphopyruvate Hydratase/metabolism , Pyloric Antrum/cytology , Pyloric Antrum/drug effects , Pyloric Antrum/metabolism , Receptors, Corticotropin-Releasing Hormone/metabolism , S100 Calcium Binding Protein G/metabolism , Serotonin/metabolism , Somatostatin/metabolism , Substance P/metabolism , Tyrosine 3-Monooxygenase/metabolism , Urocortins , Vasoactive Intestinal Peptide/metabolismABSTRACT
Actions of ANG II on electrical and synaptic behavior of enteric neurons in the guinea pig small intestine were studied. Exposure to ANG II depolarized the membrane potential and elevated neuronal excitability. The number of responding neurons was small, with responses to ANG II in 32% of submucosal neurons and 25% of myenteric neurons. Hyperpolarizing responses were evoked by ANG II in 45% of the neurons. The hyperpolarizing responses were suppressed by alpha2-noradrenergic receptor antagonists, which suggested that the hyperpolarizing responses reflected stimulation of norepinephrine release from sympathetic neurons. Exposure to ANG II enhanced the amplitude and prolonged the duration of noradrenergic inhibitory postsynaptic potentials and suppressed the amplitude of both fast and slow excitatory postsynaptic potentials. The selective ANG II(1) receptor (AT1R) antagonists, ZD-7115 and losartan, but not a selective AT2R antagonist (PD-123319), suppressed the actions of ANG II. Western blot analysis and RT-PCR confirmed expression of AT1R protein and the mRNA transcript for the AT1R in the enteric nervous system. No expression of AT2R protein or mRNA was found. Immunoreactivity for AT1R was expressed by the majority of neurons in the gastric antrum and small and large intestine. AT1R immunoreactivity was coexpressed with calbindin, choline acetyltransferase, calretinin, neuropeptide Y, and nitric oxide synthase in subpopulations of neurons. The results suggest that formation of ANG II might have paracrine-like actions in the enteric nervous system, which include alterations in neuronal excitability and facilitated release of norepinephrine from sympathetic postganglionic axons. The enhanced presence of norepinephrine is expected to suppress fast and slow excitatory neurotransmission in the enteric microcircuits and to suppress neurogenic mucosal secretion.