Localisation of NK1 receptor immunoreactivity to neurons and interstitial cells of the guinea-pig gastrointestinal tract

Tachykinins, including substance P, neurokinin A, and neuropeptides K and γ, are expressed widely in the peripheral nervous system where they affect smooth muscle contraction, exocrine gland secretion, vascular permeability, and neurotransmission. Substance P, the preferred ligand for the NK1 receptor, is found in high concentrations in the enteric nervous system. In the present study, the localisation and distribution of the NK1 receptor was studied throughout the gastrointestinal tract of the guinea-pig by using a polyclonal antiserum raised against the C-terminal 15 amino acids of the NK1 receptor. Co-localisation with other neuronal markers was examined in the ileum. Nerve cell bodies reactive for the NK1 receptor were found in the myenteric plexus of all regions and the submucous plexus of the small and large intestines. In the small intestine, the interstitial cells of Cajal were also immunoreactive. Immunoreactivity was largely confined to cell surfaces. Almost all immunoreactive myenteric nerve cells had Dogiel type I morphology, and most of these were immunoreactive for nitric oxide synthase, a transmitter of inhibitory neurons to the muscle and of descending interneurons. Neuropeptide Y-containing secretomotor neurons in the submucous and myenteric plexuses also exhibited NK1 receptor immunoreactivity. NK1 receptors were present on a minority of tachykinin immunoreactive neurons of submucous ganglia. The results suggest that receptors on the longitudinal muscle might not be conventional NK1 receptors, that excitation of the circular muscle of the ileum is indirect, perhaps via the interstitial cells of Cajal, and that enteric inhibitory neurons may be excited via NK1 receptors. © 1996 Wiley-Liss, Inc.     

Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons.

Electron microscopic immunocytochemistry was used to localize immunoreactivity for nitric oxide synthase (NOS) in whole-mount preparations of myenteric plexus and circular muscle from guinea-pig ileum. NOS immunoreactivity was patchily distributed in myenteric neurons and was not specifically associated with any subcellular organelle or with the plasma membrane. This localization leaves unanswered the question of how nitric oxide is stored and released. NOS immunoreactive fibres in the circular muscle were found closer than 100 nm to muscle cells. NOS immunoreactive nerve fibres made synaptic contacts with NOS immunoreactive and non-immunoreactive enteric neurons. These results indicate that nitric oxide may regulate the activity of both myenteric neurons and smooth muscle.     

Identification of cells that express 5- hydroxytryptamine1A receptors in the nervous systems of the bowel and pancreas

Although serotonin (5-HT)_1A receptors are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Earlier investigations have suggested that 5-HT_1A receptors are synthesized in enteric, but not pancreatic ganglia, and that they mediate pre- and postjunctional inhibition. Wholemount in situ hybridization was used to identify cells that contain mRNA encoding 5-HT_1A receptors, and immunocytochemistry was employed to locate receptor protein. mRNA encoding 5-HT_1A receptors was found in the majority of neurons in both submucosal and myenteric plexuses. 5-HT_1A immunoreactivity, however, was abundant only on the surfaces of a limited subset of nerve cell bodies and processes. 5-HT-immunoreactive axons were found in close proximity to sites of 5-HT_1A immunoreactivity. Myenteric, but not submucosal calbindin-immunoreactive neurons (with Dogiel type II morphology) were surrounded by rings of 5-HT_1A immunoreactivity. The cytoplasm of the cell bodies and dendrites of a small subset of Dogiel type I neurons was also intensely 5-HT_1A immunoreactive. Most of the Dogiel type I 5-HT_1A-immunoreactive myenteric neurons, and some of the type II neurons that were ringed by 5-HT_1A immunoreactivity became doubly labeled following injections of the retrograde tracer, FluoroGold (FG), into the submucosal plexus. 5-HT_1A immunoreactive neurons in distant submucosal ganglia also became labeled by retrograde transport of FG. None of the 5-HT_1A-immunoreactive cells were labeled by the intraluminal administration of the β-subunit of cholera toxin, a marker for vasoactive intestinal peptide-containing secretomotor neurons. These observations suggest that some of the myenteric 5-HT_1A-immunoreactive neurons project to submucosal ganglia and that the submucosal 5-HT_1A-immunoreactive cells are interneurons. In addition to neurons, a subset of 5-HT-containing enterochromaffin cells expressed 5-HT_1A immunoreactivity, which was co-localized with 5-HT in secretory granules. In the pancreas, 5-HT_1A immunoreactivity was observed in ganglia, acinar nerves, and glucagon-immunoreactive islet cells. Serotonergic enteropancreatic axons have been found to terminate in close proximity to each of these structures, which may thus be the targets of this innervation. The abundance of 5-HT_1A receptor immunoreactivity on nerves of the gut and pancreas suggests that drugs designed to interact with these receptors may have unanticipated visceral actions. © 1996 Wiley-Liss, Inc.     

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 CRF(1) receptor. CRF(1)-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.     

Neurochemically distinct classes of myenteric neurons express the mu-opioid receptor in the guinea pig ileum

The mu-opioid receptor (muOR), which mediates many of the opioid effects in the nervous system, is expressed by enteric neurons. The aims of this study were to determine whether 1) different classes of myenteric neurons in the guinea pig ileum contain muOR immunoreactivity by using double- and triple-labeling immunofluorescence and confocal microscopy, 2) muOR immunoreactivity is localized to enteric neurons immunoreactive for the endogenous opioid enkephalin, and 3) muOR immunoreactivity is localized to interstitial cells of Cajal visualized by c-kit. In the myenteric plexus, 50% of muOR-immunoreactive neurons contained choline acetyltransferase (ChAT) immunoreactivity, whereas about 43% of ChAT-immunoreactive neurons were muOR immunoreactive. Approximately 46% of muOR myenteric neurons were immunoreactive for vasoactive intestinal polypeptide (VIP), and about 31% were immunoreactive for nitric oxide synthase (NOS). MuOR immunoreactivity was found in about 68% of VIP-containing neurons and 60% of NOS-immunoreactive neurons. Triple labeling showed that about 32% of muOR neurons contained VIP and ChAT immunoreactivities. The endogenous opioid enkephalin (ENK) was observed in about 30% of muOR neurons; conversely, 48% of ENK neurons contained muOR immunoreactivity. MuOR was not detected in neurons containing calbindin, nor in interstitial cells of Cajal. MuOR-immunoreactive fibers formed a dense network around interstitial cells of Cajal in the deep muscular plexus. This study demonstrates that muOR is expressed by neurochemically distinct classes of myenteric neurons that are likely to differ functionally, is colocalized with the endogenous opioid ENK, and is not expressed by interstitial cells of Cajal.     

Colonic enteric nervous system in patients with familial amyloidotic neuropathy

The colonic enteric nervous system was investigated in autopsy specimens from 12 patients with familial amyloidotic neuropathy (FAP) and 9 controls. The infiltration of amyloid deposits in the enteric nervous system was studied by double staining for amyloid and nerve elements. The myenteric plexus was immunostained for protein gene product (PGP) 9.5, vasoactive intestinal peptide (VIP), substance P and nitric oxide synthase (NOS). The immunostained nerve elements were quantified by computerised image analysis. Double staining revealed that there was no amyloid infiltration in the ganglia, or in the nerve fibres in the colonic enteric nervous system of FAP patients. The relative volume density of PGP 9.5-immunoreactive nerve fibres in both the circular and the longitudinal muscle layers in FAP patients did not differ significantly from that of controls. The relative volume density of VIP-immunoreactive nerve fibres in the circular muscle layer was significantly decreased in FAP patients compared with controls, but not in the longitudinal layer. The number of VIP-immunoreactive neurons/mm² myenteric ganglia was significantly decreased in FAP patients. There were no statistical differences in the relative volume density for substance P- and NOS-immunoreactive nerve fibres between FAP patients and controls, nor was there any difference between FAP patients and controls regarding the number of NOS- and substance P-immunoreactive neurons/mm² myenteric ganglia. It is concluded that the colonic enteric nervous system as a whole is intact and is not damaged by amyloid infiltration. The present observation of a reduction of VIP-immunoreactive nerve fibres and neurons in myenteric plexus of FAP patients might be one of the factors that contribute to the motility disorders seen in FAP patients. Received: 1 September 1998 / Revised, accepted: 26 November 1998     

P2X2 and P2X3 receptor expression in the gallbladder of the guinea pig

We investigated for the first time, the distribution pattern of P2X2 and P2X3 receptors in the gallbladder of the guinea pig using immunohistochemistry. P2X2 and P2X3 receptor-immunoreactive nerve fibers were observed within the ganglia, in the interganglionic connectives, in the muscularis and in the paravascular plexus. Immunoreactivity for P2X2 and P2X3 was also observed in most neurons in the ganglionated plexus. Double-labeling studies revealed that 58.1% of all P2X2-positive neurons and 54.3% of all P2X3-positive neurons were found to display nitric oxide synthase. Over 90% of the neurons that were immunoreactive for P2X2 and P2X3 receptor were also immunoreactive for calretinin. We also found that 30.5% of P2X2- and 32.6% of P2X3-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide. No P2X2- or P2X3- immunoreactive neurons stained for calcitonin gene-related peptide; a few calcitonin gene-related peptide-immunoreactive nerve fibers also showed immunoreactivity to P2X2 or P2X3 receptors. These results further demonstrate the neurotransmitter diversity of the nerves of the gallbladder and provide an incentive for studies of the actions of these compounds in the gallbladder wall.     

Differential gene expression of adenosine A1, A2a, A2b, and A3 receptors in the human enteric nervous system

Adenosine receptors (ADORs) in the enteric nervous system may be of importance in the control of motor and secretomotor functions. Gene expression and distribution of neural adenosine A1, A2a, A2b, or A3 receptors (Rs) in the human intestine was investigated using immunochemical, Western blotting, RT-PCR, and short-circuit current (I(sc)) studies. Adenosine A1R, A2aR, A2bR, or A3R mRNAs were differentially expressed in neural and nonneural layers of the jejunum, ileum, colon, and cecum and in HT-29, T-84, T98G, and Bon cell lines. A1R, A2aR, A2bR, and A3R immunoreactivities (IRs) were differentially expressed in PGP 9.5-immunoreactive neurons. A2bR IR occurs exclusively in 50% of submucosal vasoactive intestinal peptide (VIP) neurons (interneurons, secretomotor or motor neurons) in jejunum, but not colon; A2aR is also found in other neurons. A3R IR occurs in 57% of substance P-positive jejunal submucosal neurons (putative intrinsic primary afferent neurons) and less than 10% of VIP neurons. Western blots revealed bands for A3R at 44 kDa, 52 kDa, and 66 kDa. A2aR and A2bR are coexpressed in enteric neurons and epithelial cells. 5'-N-methylcarboxamidoadenosine or carbachol evoked an increase in I(sc). A2bR IR is more prominent than A2aR IR in myenteric neurons, nerve fibers, or glia. A1R is expressed in jejunal myenteric neurons and colonic submucosal neurons. Regional differences also exist in smooth muscle expression of ADOR IR(s). It is concluded that neural and nonneural A1, A2a, A2b, and A3Rs may participate in the regulation of neural reflexes in the human gut. Clear cell and regional differences exist in ADOR gene expression, distribution, localization, and coexpression.     

Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity.

The bowel is the only organ of the body in which neural reflexes can be elicited in the absence of input from the brain or spinal cord. This activity is mediated by the enteric nervous system (ENS), which contains primary afferent neurons. Experiments were carried out to locate the primary afferent neurons of the ENS. Two types of stimulation were used to activate neurons in the wall of the gut in vitro: exposure of the mucosa to cholera toxin or delivery of pressure to the mucosal surface with puffs of N2 from a micropipette. Neurons that became active in response to these stimuli were identified by demonstrating the intranuclear immunoreactivity of Fos, the product of the c-fos protooncogene. No Fos immunoreactivity could be detected in the absence of stimulation; however, application of cholera toxin and puffs of N2 each induced the appearance of Fos immunoreactivity in neurons in both the submucosal and myenteric plexuses. With either stimulus, the induction of Fos immunoreactivity was antagonized by TTX and therefore depended on neuronal activity. The appearance of Fos immunoreactivity could also be prevented by the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide. In contrast, the stimulus-induced expression of Fos immunoreactivity was inhibited, but not abolished, by hexamethonium, which limited the spread of activation within the submucosal plexus and completely prevented expression of Fos immunoreactivity by myenteric neurons in response to mucosal puffs of N2. FluoroGold was injected into single ganglia of the myenteric plexus in order to identify submucosal neurons with myenteric projections. Submucosal neurons in which Fos immunoreactivity was induced by the stimuli were doubly labeled by FluoroGold. A subset of the submucosal, but not myenteric, neurons that expressed Fos immunoreactivity was doubly labeled by antibodies to calbindin. Submucosal calbindin-immunoreactive neurons were found to contain substance P immunoreactivity and could also be immunostained by anti-idiotypic antibodies that react with 5-HT1P receptors. A subset of dynorphin1-8-immunoreactive submucosal neurons (which are known to costore vasoactive intestinal peptide and to be secretomotor in function) expressed nuclear Fos immunoreactivity in response to cholera toxin, but not puffs of N2. These data suggest that intrinsic primary afferent neurons are located in the submucosal plexus, project to the myenteric plexus, and are activated by 5-HT acting on the 5-HT1P receptor subtype. These neurons are probably cholinergic and costore calbindin and substance P.     

The chemical code of porcine enteric neurons and the number of enteric glial cells are altered by dietary probiotics

Background The enteric nervous system (ENS) contains chemically coded populations of neurons that serve specific functions for the control of the gastrointestinal tract. The ability of neurons to modify their chemical code in response to luminal changes has recently been discovered. It is possible that enteric neuronal plasticity may sustain the adaptability of the gut to changes in intestinal activity or injury, and that gut neurons may respond to an altered intestinal environment by changing their neuropeptide expression. Methods We used immunohistochemical methods to investigate the presence and localization of several neuronal populations and enteric glia in both the small (ileum) and large (cecum) intestine of piglets. We assessed their abundance in submucosal and myenteric plexus from animals treated with the probiotic Pediococcus acidilactici compared with untreated controls. Key Results The treated piglets had a larger number of galanin‐ and calcitonin gene‐related peptide (CGRP)‐immunoreactive neurons than controls, but this was limited to the submucosal plexus ganglia of the ileum. Moreover, immunohistochemistry revealed that glial fibrillary acidic protein‐positive enteric glial cells were significantly higher in the inner and outer submucosal plexuses of treated animals. Conclusions & Inferences The neuronal and glial changes described here illustrate plasticity of the ENS in response to an altered luminal environment in the gastrointestinal tract.     

Intestinal infusions of oleate and glucose activate distinct enteric neurons in the rat

Nutrients entering the small intestine trigger a variety of neural and endocrine reflexes that involve specific afferents, efferents and interneurons, many of which are located within the enteric nervous system (ENS). We hypothesized that intestinal nutrient stimuli might activate specific subpopulations of these neurons. To test this hypothesis, we utilized immunohistochemical detection of nuclear c-fos expression in the myenteric and submucosal plexuses of the rat small intestine following intraintestinal infusions of oleate or glucose. Additionally, we used dual label methods to detect both Fos-immunoreactivity and immunoreactivity for five phenotypic neuronal markers: neurokinin-1 receptor (NK-1R), neurofilament-M (NF-M), neuronal nitric oxide synthase (NOS), calbindin (Cal) and calretinin (Calr), to characterize neurons that were activated by intestinal infusion of oleate and glucose. We found that oleate and glucose activated myenteric neurons in the duodenum and jejunum, but not the ileum. Oleate and glucose infusions significantly increased the number of Fos-immunoreactive nuclei in the submucosal plexus of the duodenum and jejunum, however, only glucose increased Fos-immunoreactivity in the ileum. Oleate and glucose infusions were associated with a small increase in Fos-immunoreactivity in NOS-immunoreactive neurons in the myenteric plexus. In the submucosal plexus, the majority of neurons activated by intestinal infusion of oleate or glucose were immunoreactive to Cal and Calr. In the rat, many of these neurons have Dogiel Type II-like morphology, which is consistent with the possibility that these neurons function as mucosal afferents in reflexes activated by nutrient stimuli.     

Aquaporin-4 water channels in enteric neurons

Aquaporin-4 is a water channel predominantly found in astrocytes in the central nervous system and is believed to play a critical role in the formation and maintenance of the blood-brain barrier and in water secretion from the brain. As enteric glial cells were found to share several similarities with astrocytes, we hypothesized that enteric glia might also contain aquaporin-4. We used immunohistochemistry to identify aquaporin-4 in the myenteric and submucosal plexuses of the mouse and the rat colon. We found that subpopulations of neurons in both enteric plexuses were positively labeled for human aquaporin-4. Double staining of the enteric ganglia with antibodies to the neuronal marker neurofilament-heavy chain 100 and to aquaporin-4 showed that a minority of myenteric neurons were aquaporin-4 positive (about 12% in the mouse and 13% in the rat). In contrast, in the submucosal plexus significant numbers of neurons were positive for aquaporin-4 (about 79% in both the mouse and the rat). Double labeling for aquaporin-4 and for the glial marker glial fibrillary acidic protein verified that glial cells were not immunoreactive to aquaporin-4. We further confirmed our findings with additional aquaporin-4 antibodies and Western blot analysis. We found that, in addition to expressing aquaporin-4, the myenteric plexus and, to a greater extent, the submucosal plexus both expressed aquaporin-1. We conclude that neurons rather than glial cells contain aquaporin-4 in the colonic enteric plexuses. It is known that submucosal neurons control transport processes in the intestinal mucosa, and the high percentage of aquaporin-4-positive submucosal neurons suggests that aquaporin-4 contributes to this function.     

Distribution of adrenergic receptors in the enteric nervous system of the guinea pig, mouse, and rat

Adrenergic receptors in the enteric nervous system (ENS) are important in control of the gastrointestinal tract. Here we describe the distribution of adrenergic receptors in the ENS of the ileum and colon of the guinea pig, rat, and mouse by using single- and double-labelling immunohistochemistry. In the myenteric plexus (MP) of the rat and mouse, alpha2a-adrenergic receptors (alpha2a-AR) were widely distributed on neurons and enteric glial cells. alpha2a-AR mainly colocalized with calretinin in the MP, whereas submucosal alpha2a-AR neurons colocalized with vasoactive intestinal polypeptide (VIP), neuropeptide Y, and calretinin in both species. In the guinea pig ileum, we observed widespread alpha2a-AR immunoreactivity on nerve fibers in the MP and on VIP neurons in the submucosal plexus (SMP). We observed extensive beta1-adrenergic receptor (beta1-AR) expression on neurons and nerve fibers in both the MP and the SMP of all species. Similarly, the beta2-adrenergic receptor (beta2-AR) was expressed on neurons and nerve fibers in the SMP of all species, as well as in the MP of the mouse. In the MP, beta1- and beta2-AR immunoreactivity was localized to several neuronal populations, including calretinin and nitrergic neurons. In the SMP of the guinea pig, beta1- and beta2-AR mainly colocalized with VIP, whereas, in the rat and mouse, beta1- and beta2-AR were distributed among the VIP and calretinin populations. Adrenergic receptors were widely localized on specific neuronal populations in all species studied. The role of glial alpha2a-AR is unknown. These results suggest that sympathetic innervation of the ENS is directed toward both enteric neurons and enteric glia.     

Immunohistochemical demonstration of GABA containing neurons in the guinea pig ileum using purified GABA antiserum

Antisera against GABA were prepared by immunizing rabbits with GABA conjugated to bovine serum albumin by glutaraldehyde and the antisera were then purified using a GABA immobilized epoxy-activated affinity column. Affinity chromatography and GABA-immobilized epoxy-activated agarose gels were made use of for the reduction of the cross-reactivities of GABA antiserum against endogenous amino acids. The purified GABA antiserum showed remarkably less cross-reactivity. Using this purified GABA antiserum, we noted numerous GABA-like immunoreactive (GABA-positive) nerve fibers in the myenteric meshworks and a few GABA-positive fibers exiting from the myenteric plexus of guinea pig ileum. In the myenteric ganglia, there were GABA-positive nerve cells and GABA-positive varicose fibers surrounding or running along the non-immunoreactive nerve cells. The direct visualization of enteric GABA neurons provides further support for the proposal that GABA is a neurotransmitter in the guinea pig small intestine.     

Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract.

Electrophysiologic recording and indirect immunofluorescence were combined to study localization of the medium-sized neurofilament 145 (NF145) component of the cytoskeleton in morphologically identified neurons in the myenteric and submucosal plexuses of the guinea pig enteric nervous system. Neuronal localization of chemical markers, including calbindin DK28, calretinin, nitric oxide synthase, choline-acetyltransferase, neuropeptide Y, serotonin, neurokinin 1 receptor protein, and somatostatin, was integrated with electrophysiologic and morphologic results for a more complete assessment. NF145 immunoreactivity (-IR) was present in ganglion cells with Dogiel type I morphology in the myenteric plexus of the stomach and small and large intestine. NF145-IR was not found in myenteric ganglion cells with Dogiel type II morphology. NF145-IR was not present in any of the ganglion cells in the submucosal plexus. NF145 was expressed in nerve fibers in both myenteric and submucosal plexuses. The majority of these fibers were identified as sympathetic postganglionic axons based on their disappearance in organotypic culture and on their expression of tyrosine hydroxylase. The myenteric ganglion cells with NF145-IR had electrophysiologic properties of S-type enteric neurons. NF145-IR was found in neurons with vasoactive intestinal peptide, serotonin, nitric oxide synthase, somatostatin, and neurokinin 1 receptor but not with neuropeptide Y or calbindin. The results in general suggest that NF145 is localized to distinct subsets of myenteric motor neurons and interneurons. Absence of NF145 from ganglion cells in the submucosal plexus is an example of differences between myenteric and submucosal components of the enteric nervous system.     

P2X(7) receptors in the enteric nervous system of guinea-pig small intestine.

The P2X(7) purinergic receptor subtype has been cloned and emphasized as a prototypic P2Z receptor involved in neurotransmission in the central nervous system and ATP-mediated lysis of macrophages in the immune system. Less is known about the neurobiology of P2X(7) receptors in the enteric nervous system (ENS). We studied the distribution of the receptor with indirect immunofluorescence and used selective agonists and antagonists to analyze pharmacologic aspects of its electrophysiologic behavior as determined with intracellular "sharp" microelectrodes and patch-clamp recording methods in neurons identified morphologically by biocytin injection in the ENS. Application of ATP or 2'- (or-3'-) O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzBzATP) activated an inward current in myenteric neurons. Brilliant blue G, a selective P2X(7) antagonist, suppressed the responses to both agonists. Potency of the antagonist was greatest (smaller IC(50)) for the current evoked by BzBzATP. The P2X(7) antagonists 1-[N,O-bis (1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-piperazine (KN-62) and oxidized ATP also suppressed the BzBzATP-activated current. Micropressure application of BzBzATP evoked rapidly activating depolarizing responses in intracellular studies with "sharp" microelectrodes. Oxidized-ATP suppressed these responses in both myenteric and submucosal neurons. Rapidly activating depolarizing responses evoked by application of nicotinic, serotonergic 5-HT(3), or gamma-aminobutyric acid A (GABA(A)) receptor agonists were unaffected by brilliant blue G. Immunoreactivity for the P2X(7) receptor was widely distributed surrounding ganglion cell bodies and associated with nerve fibers in both myenteric and submucous plexuses. P2X(7) immunoreactivity was colocalized with synapsin and synaptophysin and surrounded ganglion cells that contained either calbindin, calretinin, neuropeptide Y, substance P, or nitric oxide synthase. The mucosa, submucosal blood vessels, and the circular muscle coat also showed P2X(7) receptor immunoreactivity.     

Arginosuccinate synthetase, arginosuccinate lyase and NOS in canine gastrointestinal tract: immunocytochemical studies

Nitric oxide synthase (NOS) requires the substrate L ‐arginine for NO production to support multiple gastrointestinal functions. We asked, ‘Where do enzymes to regenerate L ‐arginine from L ‐citrulline exist?’. We examined loci of immunoreactivities in the canine gastrointestinal tract for arginosuccinate synthetase and arginosuccinate lyase, enzymes that resynthesize L ‐arginine from L ‐citrulline, in relation to the distribution of nNOS immunoreactivity or NADPH‐diaphorase histochemistry. Arginosuccinate synthetase and lyase were present in many neurones and nerve fibres in the myenteric plexus of the lower oesophageal sphincter (LOS), antrum, pylorus, ileum and colon; in the submucosal plexus of ileum and colon; in longitudinal muscle of ileum and colon; and in nerve bundles in circular muscle everywhere. LOS muscle was also immunoreactive for both enzymes. Circular and longitudinal muscle cells of the ileum and colon and cells resembling interstitial cells of Cajal in the deep muscular plexus of the ileum and the submuscular plexus of the colon also appeared immunoreactive. In neurones, arginosuccinate synthetase and nNOS were usually co‐localized. NADPH diaphorase activity was present in LOS and likely in pylorus, but not in muscularis externa of ileum or colon. We conclude that resynthesis of L ‐arginine probably occurs in enteric nerves, interstitial cells of Cajal (ICC) and LOS muscle; also apparently in some cells without NOS to utilize it.     

Innervation of the pancreas by neurons in the gut

Experiments were done in order to test the hypothesis that neurons in the bowel send axonal projections to the pancreas and can modify pancreatic activity. pancreatic injections of the retrograde tracer, Fluoro-Gold, labeled neurons in the myenteric plexus of the antrum of the stomach and in the first 6 cm of the duodenum. this labeling was not due to the diffusion of Fluoro-Gold from the pancreas, because the injections did not label longitudinal muscle cells overlying labeled ganglia in the bowel or neurons in the phrenic nerve nucleus or nucleus ambiguous; nor were enteric neurons labeled if insufficient time was allotted for retrograde transport. More Fluoro-Gold labeled neurons were found in the stomach (9.2 +/- 0.9/ganglion) than in the duodenum (3.8 +/- 0.3/ganglion; p less than 0.001). Neurons were found in myenteric ganglia of both duodenum and stomach that were doubly labeled by retrograde transport of Fluoro-Gold and anti-serotonin (5-HT) sera. In addition, thick bundles of 5-HT immunoreactive nerve trunks were found to run between the duodenum and the pancreas. Most 5-HT immunoreactive axons in the pancreas terminated in ganglia, although some fibers were also observed near acini, ducts, vessels, and islet cells. The B subunit of cholera toxin (B-CT) was microinjected into single myenteric ganglia in order to determine if axon terminals in the pancreas would become labeled by anterograde transport in the pancreas. B-CT labeled bundles of axons in the pancreatic stroma. Branches of these bundles entered the pancreatic parenchyma and varicose B-CT labeled terminal axons were found in pancreatic ganglia and in proximity to acinar and insulin immunoreactive cells. The intercalating fluorochrome 1, 1', dioctadecyl-3,3,3',3'-tetramethylcarbocyanine perchlorate (Dil), which moves by lateral diffusion to outline entire cells, was introduced by microinjection into individual myenteric ganglia of fixed preparations. Fluorescence was seen in sequential observations to move away from the injected ganglion along connectives of the myenteric plexus. After about a month, neurons in ganglia at some distance from the injection site displayed Dil fluorescence as did nerve bundles that exited from the myenteric plexus and pierced the longitudinal muscle in the direction of the pancreas. Varicose Dil fluorescent terminal varicosities were also observed int he pancreas. These observations indicate that there is an extensive entero-pancreatic innervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes α-bungarotoxin (α-Bgt)-insensitive nAChRs, and a polyclonal antibody raised against the α-Bgt-sensitive receptor subunit, α7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35-receptor (mAb35-R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35-R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35-R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT-immunoreactive nerve fibers. In addition, the majority of ChAT-positive neurons expressed both cell surface and internal stores of mAb35-R. In the bowel, clusters of mAb35-R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin-immunoreactive myenteric neurons. In contrast, calbindin-immunoreactive submucosal neurons did not express cell surface mAb35-R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35-R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35-R immunoreactivity was only observed in ganglia. α7-immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre- and postsynaptic nAChRs are present in the gut and pancreas. J. Comp. Neurol. 390:497–514, 1998. © 1998 Wiley-Liss, Inc.