Guo-Du Wang

Expression of type 1 corticotropin‐releasing factor receptor in the guinea pig enteric nervous system

Reverse transcription‐polymerase chain reaction (RT‐PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin‐releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT‐PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin‐filled neurons revealed that both uniaxonal neurons with S‐type electrophysiological behavior and neurons with AH‐type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF‐evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine‐30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress‐related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions. J. Comp. Neurol. 481:284–298, 2005.

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 CRF1 receptor. CRF1‐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. J. Comp. Neurol. 494:63–74, 2006.

Lubiprostone Reverses the Inhibitory Action of Morphine on Intestinal Secretion in Guinea Pig and Mouse

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 IC50 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.

Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

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.

Differential actions of urocortins on neurons of the myenteric division of the enteric nervous system in guinea pig distal colon

Background and purpose:  Urocortins (Ucns) 1, 2 and 3 are corticotropin‐releasing factor (CRF)‐related neuropeptides and may be involved in neural regulation of colonic motor functions. Nevertheless, details of the neural mechanism of action for Ucns have been unclear. We have, here, tested the hypothesis that Ucns act in the enteric nervous system (ENS) to influence colonic motor behaviour.

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 Ca²⁺ 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.

Neurogastroenterology of tegaserod (HTF 919) in the submucosal division of the guinea-pig and human enteric nervous system.

Abstract  Actions of the 5‐HT4 serotonergic receptor partial agonist, tegaserod, were investigated on mucosal secretion in the guinea‐pig and human small intestine and on electrophysiological behaviour of secretomotor neurons in the guinea‐pig small intestinal submucosal plexus. Expression of 5‐HT4 receptor protein and immunohistochemical localization of the 5‐HT4 receptor in the submucosal plexus in relation to expression and localization of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter were determined for the enteric nervous system of human and guinea‐pig small intestine. Immunoreactivity for the 5‐HT4 receptor was expressed as ring‐like fluorescence surrounding the perimeter of the neuronal cell bodies and co‐localized with the vesicular ACh transporter. Exposure of mucosal/submucosal preparations to tegaserod in Ussing chambers evoked increases in mucosal secretion reflected by stimulation of short‐circuit current. Stimulation of secretion had a relative high EC50 of 28.1 ± 1.3 μmol L−1, was resistant to neural blockade and appeared to be a direct action on the secretory epithelium. Tegaserod acted at presynaptic 5‐HT4 receptors to facilitate the release of ACh at nicotinic synapses on secretomotor neurons in the submucosal plexus. The 5‐HT2B receptor subtype was not involved in actions at nicotinic synapses or stimulation of secretion.

Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

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. J. Comp. Neurol. 511:847–862, 2008.

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.

β-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 β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-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. β-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 β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.