Guinea-pig Myenteric Plexus Research Articles

How big is the little brain in the gut? Neuronal numbers in the enteric nervous system of mice, Guinea pig, and human.

Despite numerous studies on the enteric nervous system (ENS), we lack fundamental knowledge on neuronal densities or total neuron numbers in different species. There are more anecdotal than actual figures on nerve counts. We used standardized preparation techniques and immunohistochemistry with validated panneuronal markers (human or mouse anti-HuD/C) to determine neuronal densities in specimen from the entire gastrointestinal tract of mice, guinea pig, and humans. In parallel, we measured the dimensions of the gastrointestinal regions in mouse and guinea pig. For humans, we had to rely on literature data. The average neuronal densities along the gastrointestinal tract were 35,011 ± 25,017 1/cm2 for the myenteric and 16,685 ± 9098 1/cm2 for the submucous plexus in mice, 24,315 ± 16,627 and 11,850 ± 6122 1/cm2 for guinea pig myenteric and submucous plexus, respectively, and 21,698 ± 9492 and 16,367 ± 5655 1/cm2 for human myenteric and submucous plexus, respectively. The total number of neurons in the ENS was 2.6million for mice, 14.6million for guinea pig, and 168million for human. This study reports the first comprehensive nerve cell count in mice, guinea pig, and human ENS. Neuronal densities were comparable between the three species and the differences in the total numbers of enteric neurons are likely due to body size and intestinal length. The number of enteric neurons is comparable to the number of neurons in the spinal cord for all three species.

Auto‐Immunity Targeting the Enteric Nervous System May Cause Constipation in Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that is primarily associated with somatic motor, sensory, and visual deficits. This disease can also cause gastrointestinal (GI) dysmotility, including constipation, but the etiology of this symptom in MS is poorly understood. Additionally, patients with MS typically have serum antibodies against CNS myelin and neuronal proteins that are also found in the enteric nervous system (ENS). Here, we test the hypothesis that constipation in MS is caused by auto‐antibodies that target the ENS. First, we investigated whether serum from MS patients contains antibodies that are immunoreactive against ENS targets of guinea pig myenteric plexus. We found that MS serum (n=20) targets ENS cells more intensely than age and gender matched healthy serum (n=20) (p<0.05), with stained structures including enteric neurons and glia. Next, we tested whether mice with experimental autoimmune encephalomyelitis (EAE) exhibit signs of constipation by assessing whole GI transit, colonic propulsive motility, and fecal consistency. EAE was induced in C57BL/6 or SJL/J mice by immunization against mouse spinal cord homogenate. Whole GI transit was slower in EAE SJL mice than controls (p<0.05) but was not different in EAE C57 mice. Both SJL and C57 mice with EAE had significantly slower colonic transit than control mice of the same background (C57 & SJL p<0.05). The percentage of fecal water content was significantly lower in SJL and C57 EAE mice compared to controls (C57 p<0.01, SJL p<0.0001). To test whether antibodies play a role in GI dysmotility in EAE, we assessed motility in B cell deficient EAE mice (muMt− mice). Colonic transit time was slower in EAE muMt− mice compared to muMt− control mice (p<0.05), but there was no difference in fecal water content. In summary, these data demonstrate that serum antibodies in MS patients target the ENS and could cause constipation. Further, the EAE mouse model of MS has disrupted GI motility, and this effect is partially ameliorated in B cell deficient EAE mice. Together, these data suggest that GI dysmotility in MS involves an autoimmune mediated disruption of enteric neuro‐glial function.Support or Funding InformationSupported by a pilot grant from the NMSS, and a predoctoral fellowship to ETS from NIDDK/NIH.

Retention of a new-defined intron changes pharmacology and kinetics of the full-length P2X2 receptor found in myenteric neurons of the guinea pig

P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,β-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.

The effect of ascorbate on the acetylcholine release from guinea-pig ileal myenteric plexus.

The effect of ascorbate on the release of acetylcholine from the longitudinal muscle-myenteric plexus preparation of the guinea-pig isolated ileum has been investigated using a bioassay and an isotopic technique. Ascorbate at 5 mM increased the spontaneous output of acetylcholine and enhanced DMPP-induced output of acetylcholine, while iso-ascorbate, 5 mM, did not. Ascorbate did not influence either the spontaneous or the DMPP-induced release of acetylcholine from synaptosomes of the ileal myenteric plexus. These results suggest that ascorbate promotes acetylcholine release from intramural cholinergic nerves.

Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds.

Quantitative assessment of glial cells in the human and guinea pig enteric nervous system with an anti‐Sox8/9/10 antibody

Quantitative changes of enteric glia (EGC) have been implicated in gastrointestinal disorders. To facilitate future studies of EGC in human pathology, we aimed to characterize thoroughly glial markers in the human enteric nervous system (ENS) and to compare EGC in man and guinea pig. Whole-mount preparations of the enteric nerve plexuses from human and guinea pig ileum and colon were labeled with antibodies against S100b, glial fibrillary acidic protein (GFAP), and p75NGFR and the transcription factors Sox8/9/10 and neuronally counterstained. Abundant immunoreactivity (IR) for S100b, GFAP, p75NGFR, and Sox8/9/10 was detected in EGC of all studied regions. Although the cytoplasmatic staining pattern of most markers did not permit glial quantification, the nuclear localization of Sox8/9/10-IR allowed to identify and count all EGC individually. In both man and guinea pig, myenteric ganglia were larger and contained more EGC and neurons than submucous ganglia. Furthermore, there were more EGC in the human than in the guinea pig myenteric plexus (MP), glial density was consistently higher in the human ENS, and the glia index (glia:neuron ratio) ranged from 1.3 to 1.9 and from 5.9 to 7.0 in the human submucous plexus (SMP) and MP, respectively, whereas, in guinea pig, the glia index was 0.8-1.0 in the SMP and 1.7 in the MP. The glia index was the most robust quantitative descriptor within one species. This is a comprehensive set of quantitative EGC measures in man and guinea pig that provides a basis for pathological assessment of glial proliferation and/or degeneration in the diseased gut.

Functional interaction between α 2-adrenoceptors, μ- and κ-opioid receptors in the guinea pig myenteric plexus: Effect of chronic desipramine treatment

The existence of a functional interplay between α 2-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to α 2-adrenoceptor, μ- and κ-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of α 2-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric μ- and κ-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively α 2A-adrenoceptor and κ-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), μ-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of α 2A-adrenoceptors, μ- and κ-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of α 2A-adrenoceptors, μ- and κ-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory G i/o proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory G i/o proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the μ-opioid agonist might compensate for the development of α 2A-adrenoceptor and κ-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of α 2-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for μ-opioid, but not for κ-opioid receptors.

Actions of orexins on individual myenteric neurons of the guinea-pig ileum: orexin A or B?

The actions of orexins (orexin A and B, 10-300 nM) on individual myenteric neurons of the guinea-pig ileum in vitro were compared using intracellular recording methods. Both orexins caused membrane depolarizations associated with an increase in input neuronal resistance in S and AH neurons via a direct action. Orexin depolarizations reversed at about -90 mV, indicating they were due to an inactivation of K+ channels. Orexins facilitated fast excitatory postsynaptic potentials without affecting postsynaptic sensitivity to acetylcholine and adenosine 5'-triphosphate, indicating that the peptides may facilitate ganglionic transmission by increasing presynaptic release of neurotransmitters. Orexin B was sometimes more effective than orexin A and vice versa. It is concluded that orexin B increased neuronal activity via mechanisms similar to orexin A in the guinea-pig myenteric plexus.

Human mast cell mediator cocktail excites neurons in human and guinea‐pig enteric nervous system

Neuroimmune interactions are an integral part of gut physiology and involved in the pathogenesis of inflammatory and functional bowel disorders. Mast cells and their mediators are important conveyors in the communication from the innate enteric immune system to the enteric nervous system (ENS). However, it is not known whether a mediator cocktail released from activated human mast cells affects neural activity in the ENS. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human ENS after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor cross-linking. Local application of MCMC onto individual ganglia evoked an excitatory response consisting of action potential discharge. This excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Compound action potentials from nerve fibres or fast excitatory synaptic inputs were not affected by MCMC. This study demonstrates immunoneural signalling in the human gut and revealed for the first time that an MCMC released from stimulated human intestinal mast cells induces excitatory actions in the human and guinea-pig ENS.

Human mast cell mediators excite neurones in murine and human enteric nervous system

Neuroimmune interactions are an integral part of gut pyhsiology and involved in the pathogenesis of inflammatory as well as functional bowel disorders. Mast cells and their mediators are pivotal to communicate information from the innate enteric immune system to the enteric nervous system. So far it is not known whether a mediator cocktail released from activated human mast cells affect neural activity and whether mast cell mediators have effects on human enteric nerves. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human enteric nervous system after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor crosslinking. We studied 106 nerve cells from 8 human submucous plexus preparations, 64 nerve cells from 9 guinea-pig submucous plexus preparations and 1095 nerve cells from 4 guinea-pig myenteric plexus preparations. Local application of MCMC for 500–100ms onto individual ganglia evoked an excitatory response consisting of action potential discharge. This postsynaptic excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Fast excitatory synaptic inputs were not affected by MCMC indicating that MCMC had no presynaptic action. This study revealed for the first time that a mast cell mediator cocktail released from stimulated human intestinal mast cells induces excitatory actions in the human and murine enteric nervous system. We thereby present evidence for the concept that immunoneural signals arising from mast cells are able to enhance activity in the enteric nervous system.

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus

Supersensitivity to muscarinic, κ- and μ-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (κ-opioid receptor agonist) and DAMGO (μ-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, κ- and μ-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas κ- and μ-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Localization of a functional GABA-A receptor in the guinea pig myenteric plexus

Localization of a functional GABA-A receptor in the guinea pig myenteric plexus

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.

Vanilloid receptor type 1-immunoreactivity is expressed by intrinsic afferent neurones in the guinea-pig myenteric plexus

The enteric sensory nervous system consists of extrinsic and intrinsic components. The cellular distribution of vanilloid receptor type 1 (VR1) and its relation to the intrinsic sensory neurones were studied in myenteric plexus-longitudinal muscle preparations of rat ileum and guinea-pig ileum and colon. VR1-immunoreactivity was localized on fine fibres and expressed by ganglionic cells. In the guinea-pig myenteric plexus, a proportion of VR1-immunoreactive cells co-localized with calbindin, a marker for intrinsic afferent neurones. Reverse transcription-polymerase chain reaction with rat VR1-specific primers detected VR1 mRNA in rat but not in guinea-pig preparations. We conclude that VR1 is expressed on fibres and by myenteric neurones. In the guinea-pig, VR1 is expressed by intrinsic afferent neurones but its mRNA may differ from the rat sequence in the region of the primers.

Distribution and chemical coding of orphanin FQ/nociceptin-immunoreactive neurons in the myenteric plexus of guinea pig intestines and sphincter of Oddi.

Longitudinal muscle-myenteric plexus preparations of guinea pig intestines and sphincter of Oddi (SO) were immunostained for orphanin FQ/nociceptin. Orphanin FQ-immunoreactive (OFQ-IR) neurons and nerve fibers were relatively abundant in the SO, duodenum, ileum, cecum, and distal colon, with fewer neurons and nerve fibers observed in the proximal colon. Double staining with antibodies directed against the neuron-specific RNA binding protein Hu revealed that while the numbers of OFQ-IR neurons per ganglion decreased along the gut tube, similar proportions (7-9%) of neurons in these regions were OFQ-IR, whereas <1% of the neurons in the proximal colon were OFQ positive. In the ileum, where 8% of the myenteric neurons were OFQ-IR, all OFQ-IR neurons expressed choline acetyltransferase. In addition, multiple-label immunohistochemistry demonstrated that 58% of the OFQ-IR neurons were calretinin-IR, 52% were substance P-IR, and 28% were enkephalin-IR. Nitric oxide synthase immunoreactivity was observed in about 5% of OFQ-IR neurons, or 0.4% of the total population, and a similar proportion of the OFQ-IR neurons was positive for vasoactive intestinal peptide. No OFQ-IR neurons were immunoreactive for calbindin, somatostatin, or serotonin. These results, combined with previous studies of chemical coding and projection patterns in the guinea pig myenteric plexus, indicate that OFQ-IR is expressed preferentially in excitatory motor neurons projecting to the longitudinal and circular muscle layers, as well as a small subgroup of descending interneurons. Because OFQ is expressed by excitatory motor neurons, and because this peptide inhibits excitatory neurotransmission in the guinea pig ileum, it is likely that OFQ acts through a feedback autoinhibitory mechanism.

Visualization of nitric oxide formation in cell cultures and living tissue.

We have visualized nitric oxide (NO) released from cell cultures and living tissue. NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera. Murine macrophages were activated with interferon-gamma (IFN-gamma) and endotoxin (LPS). Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea-pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated. There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation. The stimulation-induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L-arginine from the medium also decreased photon counts. The present method allowed integration times in the order of minutes to improve signal-to-noise ratio. However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films. Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors. Thus, this method has demonstrated specificity for the L-arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution.

GABAergic inhibition of nitric oxide-mediated relaxation of guinea-pig ileum.

The effects of GABA receptor agonists were investigated on guinea-pig isolated ileum longitudinal muscle with intact myenteric plexus. Electrical field stimulation (1 Hz, 10 s) of the histamine (1 microM)-precontracted preparation caused a contraction followed by a relaxation. Relaxations were inhibited by L-N(G)-nitroarginine (L-NA; EC50 3 microM) in a concentration-dependent manner. The inhibitory action of 10 microM L-NA was blocked by 10 microM L-arginine but not by D-arginine, which indicates that the relaxation was largely mediated by endogenous nitric oxide (NO). Tetrodotoxin (1 microM) reduced the relaxation only by about 50%. GABA and the GABA(B) agonist, baclofen, inhibited the field stimulation-induced longitudinal muscle relaxation in a concentration-dependent manner. The GABA(B) receptor antagonist, saclofen (10 microM), antagonised the effect of baclofen (apparent pA2 of saclofen: 5.6). Muscimol (10 microM) similarly inhibited the relaxation, and this inhibition was prevented by bicuculline (1 microM). It is concluded that nitrergic nerves of the guinea-pig myenteric plexus are endowed with GABA(A) and GABA(B) receptors which mediate inhibition of NO release.

Evidence for a neurotransmitter role of glutamate in guinea pig myenteric plexus neurons

Longitudinal muscle-myenteric plexus strips of the guinea pig ileum were used to investigate a possible transmitter role of glutamate (Glu) in the enteric nervous system. Glu was released from this nerve muscle preparation by high K +-depolarization in a Ca 2+-dependent manner, by electrical field stimulation and by the ganglionic stimulant dimethylphenylpiperazinium which indicates its neural origin. Contractions of the longitudinal muscle evoked by electrical stimulation of the myenteric nerves or by Glu, were significantly reduced by the N-methyl- d-aspartate (NMDA)-receptor antagonist FR115427 (9 and 18 μM), whereas contractions induced by histamine were unaffected. The results show that the amino acid Glu is likely to play an excitatory neurotransmitter role via NMDA receptors in the myenteric plexus of the guinea pig.

Role of protein kinase C in the release of [ 3H]acetylcholine from myenteric plexus treated with vesamicol

The present experiments investigated the release of [ 3H]acetylcholine ([ 3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na +-K + ATPase inhibitor, released [ 3H]ACh synthesised in the presence of (−)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca 2+-dependent and in the presence of (−)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (−)-vesamicol from interfering with the release of [ 3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [ 3H]ACh induced by ouabain in the presence of (−)-vesamicol depends on PKC activation.

Selectivity of hemicholinium mustard, an affinity ligand, for the high-affinity choline transport system

The selectivity of the irreversible inhibition of high-affinity choline uptake (HACU) by hemicholinium mustard (HCM; 2,2′-(4,4′-biphenylene)bis[2-hydroxy-4-(2-bromoethyl)-morpholine] hydrochloride) with respect to other cholinergic proteins and other sodium-dependent transport systems was examined. Preincubation of rat forebrain membranes with HCM, followed by washing and measurement of [ 3H]-hemicholinium-3 binding to the high-affinity choline transporter, was shown to decrease binding capacity (B max) by 70% without affecting the apparent affinity of the ligand. However, a similar preincubation, wash and binding experiment using [ 3H]-NMS as a ligand for muscarinic receptors showed no HCM effect on binding parameters. To measure the effects of HCM on choline acetyltransferase (ChAT), synaptosomes were incubated in HCM, then washed. The synaptosomes were lysed and ChAT activity was measured. Treatment with 50 μM. HCM, a concentration that inhibits 100% of synaptosomal HACU, results in a 24% decrease in ChAT activity. HCM demonstrates little residual inhibition of other sodium-dependent neurotransmitter transporter transporters: preincubation with 50 μM HCM results in a decrease of 12% in transport of [ 3H]-dopamine and a decrease of 6% in the transport of [ 3H]-GABA. The binding of HCM, like that of hemicholinium-3 is sodium-dependent. HCM preincubation in the presence of sodium results in inhibition of HACU to 32% of control; in the absence of sodium HACU is 65% of control. This represents a loss of 51% of the observed irreversible inhibition produced by HCM. Irreversible inhibition by HCM can also be prevented by co-incubation with hemicholinium-3. Co-incubation with hemicholinium-3 results in residual HACU inhibition that decreases from 51% (HCM alone) to 28% (HCM + hemicholinium-3). When atropine instead of hemicholinium-3 is co-incubated with HCM, HCM still inhibits 40% of transport, demonstrating the pharmacological specificity of the protective effect of hemicholinium-3. Experiments in the guinea-pig myenteric plexus preparation demonstrate a gradual recovery from the residual effects of HCM. Evoked ACh release decreases to 24% immediately following treatment with 1 μM HCM. After 2 hr of recovery, tissues have recovered to about 50% of control levels, after which recovery continues at a slower rate.