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.     

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.     

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.     

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.     

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.     

Failure to demonstrate serotonergic neurons in the myenteric plexus of the rat

Summary Several recent studies suggested that serotonergic neuron-like elements are present in the guinea pig ileum. The present paper reports an extensive study of the digestive tract of the rat with the use of a histofluorescence technique. Administration of the serotonin precursor, tryptophan, associated with a monoamine oxidase inhibitor, did not allow histochemical demonstration of rapidly fading, yellow fluorescent, 6-hydroxydopamine-resistant neurons; conversely such neurons were readily detected in the brain. It is concluded that serotonergic neuron-like elements cannot be detected histochemically in the rat myenteric plexus area after chemical sympathectomy.     

Neuropeptides in the gut: quantification and characterization of cholecystokinin octapeptide-, bombesin- and vasoactive intestinal polypeptide-like immunoreactivities in the myenteric plexus of the guinea-pig small institute

The localization of CCK8-, bombesin- and VIP-like immunoreactivities in the myenteric plexus of the guinea pig small intestine has been studied by radioimmunoassay of extracts of longitudinal muscle strips obtained with and without adherent myenteric plexus; concentrations were compared with those in other regions of the gut. In innervated strips of longitudinal muscle of ileum there was approximately 14 pmol/g CCK8-, 32 pmol/g bombesin- and 135 pmol/g VIP-like immunoreactivity; concentrations were reduced by over 70% in denervated strips. Gel filtration and ion exchange chromatography indicated that over 80% of CCK immunoreactivity was due to CCK8; no evidence was found of significant amounts of smaller COOH-terminal fragments. Bombesin immunoreactivity occurred in two forms, the major one resembling the amphibian tetradecapeptide in its elution from gel filtration columns. Immunoreactive VIP differed markedly from porcine VIP in immunochemical and chromatographic properties; the data suggest that guinea pig VIP is less basic than porcine VIP and that the two peptides differ in structure in their NH2-terminal regions. Some functional implications of these findings are discussed.     

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

5-HT4 receptor activation facilitates recovery from synaptic rundown and increases transmitter release from single varicosities of myenteric neurons

5-HT(4) receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the 5-HT(4) receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEPSPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 +/- 0.1 s) from 17 +/- 2 mV to 0.7 +/- 0.3 mV. Renzapride (0.1 microM) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 +/- 2 s to 1.6 +/- 0.2 s (P < 0.05). 5-HT (0.1 microM) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 muM), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 microM) increased release probability twofold. We conclude that 5-HT(4) receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of 5-HT(4) receptor agonists.     

Quinoxalin-2-carboxamides: synthesis and pharmacological evaluation as serotonin type-3 (5-HT3) receptor antagonists

A series of quinoxalin-2-carboxamides were designed as per the pharmacophoric requirements of 5-HT(3) receptor antagonists and synthesized by condensing the carboxylic group of quinoxalin-2-carboxylic acid with various amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The structures of the synthesized compounds were confirmed by physical and spectroscopic data. The carboxamides were evaluated for their 5-HT(3) receptor antagonisms in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-HT(3) agonist, 2-methy-5-HT. All the synthesized compounds showed 5-HT(3) receptor antagonism, (4-benzylpiperazin-1-yl)(quinoxalin-2-yl)methanone was the most potent compound among this series.     

Immunohistochemical identification of serotonin and noradrenalin containing structures within the nerve plexuses of rat ileum

The distribution of 5-hydroxytryptamine (= serotonin = 5-HT) and noradrenalin (NA) in the enteric plexuses of the rat ileum was studied using immunocytochemical techniques. 5-HT-like immunoreactive fibers were observed only in the myenteric plexus, surrounding the ganglionic cells, which are all unreactive. NA-like immunoreactive fibers were present in all layers of the ileum: in the myenteric plexus, they were localized in the nodes, forming a network all round the neuronal perikarya; in the Meissner plexus, positive axons were arranged in a delicate network; submucosal blood vessels were often provided by NA-immunopositive nerve plexus. In the inner circular muscle layer the immunoreactive NA-positive fibers run within nerve bundles mainly parallel with the smooth muscle cells. The 5-HT immunoreactive material was depleted by treatment with reserpine; depletion of NA by 6-hydroxy-dopamine was also observed; on the contrary, no depletion of 5-HT by 5,7-dihydroxytryptamine was obtained. To confirm the validity of these results, specific antibodies to tyrosine hydroxylase (TH) and aromatic 1-aminoacid-decarboxylase (AADC), two enzymes involved in the synthesis of catecholamines, were used. In conclusion these experiments indicate that 5-HT is present, probably as a transmitter, in certain fibres of the rat myenteric plexus, distributed in a way similar to that of NA-containing fibers. However, at variance with NA fibers, 5-HT fibers are not present in other regions of the intestine wall.     

Differential localization of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the human rectum

The functions of the 5-hydroxytryptamine3 (5-HT3) and 5-hydroxytryptamine4 (5-HT4) receptors in gastrointestinal tract are complex depending on the species and anatomical regions, and the localization of these receptors in the human rectum was unclear. We examined the localization of the 5-HT3 and 5-HT4 receptors in human rectum by in vitro receptor autoradiography using [125I](S)iodozacopride and [125I] SB207710 as a ligand, respectively. Specific [125I](S)iodozacopride binding sites were clearly evident in the myenteric plexus, whereas, low levels of [125I]SB207710 binding sites were distributed over the muscle but not to the myenteric plexus. The 5-HT3 receptor located on the myenteric plexus and the 5-HT4 receptor on the smooth muscle may participate in contractility and relaxation of human rectum, respectively.     

Effects of narcotic opiates and serotonin on the electrical behavior of neurons in the guinea pig myenteric plexus

Action potentials were recorded extracellularly from spontaneously firing neurons in the myenteric plexus of the guinea pig ileum. Morphine, which inhibits acetylcholine release from the myenteric plexus, inhibited the spontaneous electrical activity of about half the cells studied, while serotonin elevated the firing rate of these cells. Units not stimulated by serotonin were not inhibited by morphine or levorphanol. Morphine also prevented the increase in firing rate caused by serotonin. These effects of morphine were stereospecific and blocked by naloxone, and are therefore considered to be specific opiate effects. This study demonstrates opposing effects of narcotic opiates and serotonin on the electrical activity of serotoninoceptive neurons in the myenteric plexus.     

Autoradiographic localization of binding sites for galanin and VIP in small intestine

The distribution of the binding sites for [125I]galanin and [125I]iodotyrosyl VIP in sections of small intestine from rat, rabbit, guinea pig and man was investigated using in vitro receptor autoradiography. The specific binding sites for [125I]galanin were most concentrated in the myenteric plexus and longitudinal muscle layers. The distribution of [125I]VIP binding was similar, with the highest level of binding in the myenteric plexus. Some VIP binding was also seen in the mucosa. The abundance of binding sites for both peptides in the myenteric plexus suggests that this is the anatomical site of the functional antagonism between these endogenous peptides in the mammalian intestine.     

Single neuron studies of opiate action in the guinea pig myenteric plexus.

The action of narcotics and other drugs on electrical activity of neurons in the guinea pig myenteric plexus was examined by extracellular recording with a suction electrode. Morphine, in a stereospecific and naloxone-sensitive action, inhibits spontaneous electrical activity of many neurons, and antagonizes an increased firing rate caused by serotonin or nicotine. The inhibition by morphine of spontaneous electrical activity occurs under conditions of synaptic transmission blockade, which renders unlikely several possible synaptic mechanisms in the primary effect of opiates. Morphine was found not to alter conduction velocity of myenteric neurons. It is concluded that morphine probably acts to reduce the excitability of a class of myenteric plexus neurons, perhaps by hyperpolarizing or stabilizing the membrane potential.     

An Artifact Of Liquid Emulsion Autoradiography

The localization of ³H-opiatcs in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ilial sections to dried photographic emulsion avoids this source of error.     

TRH-like immunoreactivity in endocrine cells and neurons in the gastro-intestinal tract of the rat and guinea pig

Summary By use of the indirect immunofluorescence technique, the cellular localization of thyrotropin-releasing hormone (TRH) was studied in the gastrointestinal tract of rats and guinea pigs of different ages. TRH-like immunoreactivity (LI) was observed in many pancreatic islet cells of young rats and guinea pigs but only in single cells of 6-month-old rats. In aged guinea pigs, a reduction in the number of TRH-positive cells was evident; however, numerous strongly fluorescent cells were still present. In the guinea pig, TRH-LI was in addition observed in gastrin cells in the stomach. TRH-positive nerve fibers occurred in the myenteric plexus of the oesophagus, stomach and intestine of the rat, and in the muscle layers of the guinea pig. These results suggest a functional role of TRH both as hormone and neuroactive compound in various portions and sites of the gastro-intestinal tract of the rat and guinea pig     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.