Effect of motilin on the contractility of gastric smooth muscle via NO pathway in guinea pigs

This study investigates the gastroprokinetic effects of motilin and erythromycin A (EM-A) and its potential mechanism in guinea pigs Cavia porcellus in vitro. Guinea pig stomach strips were mounted under organ baths containing Krebs solution. Motilin,EM-A,Nω-Nitro-L-arginine (L-NNA),L-arginine (L-AA) were added to the bathing solution in a non-cumulative way. Then the effects of motilin and EM-A was studied during electrical field stimulation (EFS) in the absence and presence of L-NNA and L-AA in the gastri...     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

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.     

Oxytocin is expressed by both intrinsic sensory and secretomotor neurons in the enteric nervous system of guinea pig

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.     

Interdigestive migrating contractions are coregulated by ghrelin and motilin in conscious dogs

During fasting, gastrointestinal (GI) motility is characterized by cyclical motor contractions. These contractions have been referred to as interdigestive migrating contractions (IMCs). In dogs and humans, IMCs are known to be regulated by motilin. However, in rats and mice, IMCs are regulated by ghrelin. It is not clear how these peptides influence each other in vivo. The aim of the present study was to investigate the relationship between ghrelin and motilin in conscious dogs. Twenty healthy beagles were used in this study. Force transducers were implanted in the stomach, duodenum, and jejunum to monitor GI motility. Subsequent GI motility was recorded and quantified by calculating the motility index. In examination 1, blood samples were collected in the interdigestive state, and levels of plasma ghrelin and motilin were measured. Plasma motilin peaks were observed during every gastric phase III, and plasma ghrelin peaks occurred in nearly every early phase I. Plasma motilin and ghrelin levels increased and decreased cyclically with the interdigestive states. In examination 2, saline or canine ghrelin was administered intravenously during phase II and phase III. After injection of ghrelin, plasma motilin levels were measured. Ghrelin injection during phases II and III inhibited phase III contractions and decreased plasma motilin levels. In examination 3, ghrelin was infused in the presence of the growth hormone secretagogue receptors antagonist [D-Lys3]-GHRP-6. Continuous ghrelin infusion suppressed motilin release, an effect abrogated by the infusion of [D-Lys3]-GHRP-6. Examination 4 was performed to evaluate the plasma ghrelin response to motilin administration. Motilin infusion immediately decreased ghrelin levels. In this study, we demonstrated that motilin and ghrelin cooperatively control the function of gastric IMCs in conscious dogs. Our findings suggest that ghrelin regulates the function and release of motilin and that motilin may also regulate ghrelin.     

Chemical coding of neurons expressing δ- and κ-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.     

Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus

Motilin and ghrelin are the gastrointestinal (GI) hormones released in a fasting state to stimulate the GI motility of the migrating motor complex (MMC). We focused on coordination of the ghrelin/motilin family in gastric contraction in vivo and in vitro using the house musk shrew (Suncus murinus), a ghrelin- and motilin-producing mammal. To measure the contractile activity of the stomach in vivo, we recorded GI contractions either in the free-moving conscious or anesthetized S. murinus and examined the effects of administration of motilin and/or ghrelin on spontaneous MMC in the fasting state. In the in vitro study, we also studied the coordinative effect of these hormones on the isolated stomach using an organ bath. In the fasting state, phase I, II, and III contractions were clearly recorded in the gastric body (as observed in humans and dogs). Intravenous infusion of ghrelin stimulated gastric contraction in the latter half of phase I and in the phase II in a dose-dependent manner. Continuous intravenous infusion of ghrelin antagonist (d-Lys3-GHRP6) significantly suppressed spontaneous phase II contractions and prolonged the time of occurrence of the peak of phase III contractions. However, intravenous infusion of motilin antagonist (MA-2029) did not inhibit phase II contractions but delayed the occurrence of phase III contractions of the MMC. In the in vitro study, even though a high dose of ghrelin did not stimulate contraction of stomach preparations, ghrelin administration (10(-10)-10(-7) M) with pretreatment of a low dose of motilin (10(-10) M) induced gastric contraction in a dose-dependent manner. Pretreatment with 10(-8) M ghrelin enhanced motilin-stimulated gastric contractions by 10 times. The interrelation of these peptides was also demonstrated in the anesthetized S. murinus. The results suggest that ghrelin is important for the phase II contraction and that coordination of motilin and ghrelin are necessary to initiate phase III contraction of the MMC.     

Localization of CB1-cannabinoid receptor immunoreactivity in the porcine enteric nervous system

Cannabis has been used for centuries in the medicinal treatment of gastrointestinal disorders. Endogenous cannabinimimetic substances such as 2-arachidonylglycerol have been isolated from gut homogenates and CB1-cannabinoid binding sites have been identified in small intestine. In this study, CB1-cannabinoid receptors (CB1-R) were immunohistochemically localized within the enteric nervous system of the pig, an omnivorous species whose digestive tract is functionally similar to humans. Two anti-CB1-R antisera, raised against N-terminal epitopes in the human CB1-R, were employed to localize receptor immunoreactivity by secondary immunofluorescence. CB1-R immunoreactivity was observed in the myenteric and submucosal ganglionated plexuses of porcine ileum and colon. In the ileum, all CB1-R-immunoreactive neurons coexpressed immunoreactivity to the cholinergic marker, choline acetyltransferase (ChAT). CB1-R/ChAT-immunoreactive neurons appeared to be in close apposition to ileal Peyer's patches, submucosal blood vessels, and intestinal crypts. In the distal colon, CB1-R-immunoreactive neurons also expressed immunoreactivity to ChAT, albeit less frequently than in ileum. Immunoreactivity to vasoactive intestinal peptide or nitric oxide synthase was not colocalized in ileal or colonic CB1-R-immunoreactive neurons. These studies indicate that CB1-R are present in cholinergic neurons in the porcine enteric nervous system. The potential roles of these receptors in intestinal motility and epithelial transport, host defense and visceral pain transmission are discussed.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Differential distribution of 5-hydroxytryptamine3 receptor in the colon between human and guinea pig

Localization of 5-hydroxytryptamine3 (5-HT3) receptor in the human colon was examined by in vitro receptor autoradiography using [125I](S)iodozacopride, and compared with that in the guinea pig colon. [125I](S)iodozacopride binding sites were found with high densities around the myenteric plexus, but with low ones in the muscle layer and mucosa of the human colon, and the binding was abolished by granisetron, a specific 5-HT3 receptor antagonist. While in the guinea pig colon, specific [125I](S) iodozacopride binding was not detected in either the myenteric plexus or the muscle layers. Thus, the 5-HT3 receptors are present in the human colon, especially densely located in the myenteric plexus, but not in the guinea pig colon, and those may participate in the colonic motility. The results of functional studies of 5-HT3 receptor obtained from experiments using guinea pig are not always applying to the human.     

Physiological analysis of delta opioid receptors in the guinea pig myenteric plexus

Ilea taken from guinea pigs that had been chronically exposed to morphine exhibit a greater tolerance to morphine and normorphine than to the opioid peptides D-ala2-D-leu5-enkephalin (DADLE) or D-met2-pro5-enkephalinamide (DMPE). This differential tolerance strongly implies the existence of at least two different types of opioid receptor in the guinea pig myenteric plexus or two different mechanisms of interaction between opioids and their receptor complex. Since DADLE is considered to be the prototypic ligand for the delta receptor, the above results imply the presence of delta receptors in the guinea pig myenteric plexus and furthermore, that this subtype of opioid receptor is associated with the modulation of release of enteric acetylcholine.     

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Sympathetic denervation-induced changes in G protein expression in enteric neurons of the guinea pig colon

Chronic sympathetic denervation entails subsensitivity to alpha(2)-adrenoceptor agonists and supersensitivity to kappa- and mu-opioid receptor agonists modulating cholinergic neurons in the guinea pig colon. A possible role for signal transduction G proteins in contributing to development of these sensitivity changes was investigated. Pertussis toxin (PTX), a blocker of the G(i/o)-type family of G proteins significantly reduced the inhibitory effects of UK14,304 (alpha(2)-adrenoceptor agonist), U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on acetylcholine (ACh) overflow in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. In this experimental condition, immunoblot analysis revealed reduced levels of G(alphao), G(alphai2), G(alphai3) and G(beta) in myenteric plexus synaptosomes. On reverse, synaptosomal levels of G(alphai1) and G(alphaz), a PTX-insensitive G-protein, increased after chronic ablation of the sympathetic pathways. These data suggest that changes in the function and expression of inhibitory G proteins coupled to alpha(2)-adrenoceptors, kappa- and mu-opioid receptors occur in the myenteric plexus of the guinea pig colon after chronic sympathetic denervation. The possibility that regulation of G proteins represents one of the biochemical mechanisms at the basis of the changes in sensitivity of enteric cholinergic neurons to alpha(2)-adrenoceptor, kappa- and mu-opioid receptor agonists is discussed.     

Age-related changes in the gastrointestinal tract: a functional and immunohistochemical study in guinea-pig ileum

It is known that there is an age-related increase in gastrointestinal diseases. However, there is a lack of studies dealing with the correlation between age-related changes in function and intrinsic innervation in the gastrointestinal tract. The purpose of this work was to study this subject in the guinea pig ileum, whose functional and structural features are well known in the young age. Ileal longitudinal muscle — myenteric plexus (LMMP) preparations were obtained from 3-to 24-month-old guinea pigs. Both functional and immunohistochemical techniques were applied. The force of the contraction elicited by excitatory stimuli (electrical stimulation, acetylcholine, substance P, and opioid withdrawal) increased in parallel with an age-dependent reduction in the density of excitatory motor neurones to the longitudinal muscle, whereas other subpopulations of neurones, including inhibitory motor neurones, decreased much more slowly. Although the increase in responsiveness could be related to the age/weight-related increment in muscle bulk, some compensatory modifications to the lowered density of excitatory neurones could also be involved. On the other hand, the acute inhibitory response to morphine remained unaltered in old animals, whilst in vitro tolerance was lower. These results suggest that although age-dependent neuronal loss does not cause dramatic changes in intestinal motility, it is a factor that could contribute to disturbing normal responsiveness and, perhaps, underlie the higher frequency of gastrointestinal diseases encountered in the elderly.     

CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.     

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.