Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998     

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species

Summary Calcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.     

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.     

The distribution of an immunoreactive endothelin-like substance in the nervous system of the nereid,Neanthes diversicolor (Annelida)

Summary The distribution of an immunoreactive endothelin-1-like peptide was investigated in the nereid, Neanthes diversicolor, using an antiserum raised against synthetic endothelin-1. Immunoreactive perikarya were localized in the brain, and nerve fibers containing endothelin-1-like material were found in the neuropil occupying the central portion of the brain. No immunostained fiber elements were traced in the circumesophageal connectives. Immunoreactive perikarya occurred in the subesophageal ganglion. From this ganglion, specifically stained fibers run posteriorly toward the ventral nerve cord. In each segmental ganglion, immunoreactive neurons were observed in medio-ventral and latero-ventral regions, and one or two marked fibers extended to the parapodium. In the parapodium, small immunoreactive perikarya and fiber elements were visible. Immunolabeled fibers occurred in the stomatogastric nerves, in the wall of the buccal cavity, and in the pharynx, esophagus, intestine and its anal region. Immunoreactive perikarya and nerve fibers were visualized between the circular muscle layer and epithelial cell layer in the esophagus and intestine. The endothelin-1-like substance shown to occur in N. diversicolor appears to function as a neurotransmitter or neuromodulator.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

An immuno-electron-microscopic study of the localization of VIP-like immunoreactivity in the adrenal gland of the rat

Summary VIP-like immunoreactivity was revealed in a few chromaffin cells, medullary ganglion cells and a plexus of varicose nerve fibers in the superficial cortex and single varicose fibers in the juxtamedullary cortex and the medulla of the rat adrenal gland. VIP-like immunoreactive chromaffin cells were polygonal in shape without any distinct cytoplasmic processes and they appeared solitarily. Their cytoplasm contained abundant granular vesicles having a round core and the immunoreactive material was localized to the granular core. VIP-immunoreactive ganglion cells were multipolar and had large intracytoplasmic vacuoles. The immunoreactive material was localized not only in a few granular vesicles but also diffusely throughout the axoplasm. VIP-immunoreactive varicose nerve fibers in the superficial cortex were characterized by abundant small clear vesicles and some large granular vesicles, while those in the juxtamedullary cortex and medulla and the ganglionic processes were characterized by abundant large clear vesicles, as well as the same vesicular elements as contained in the nerves in the superficial cortex. The immunoreactive material was localized on the granular cores and diffusely in the axoplasm in both nerves. Based on the similarity and difference in the composition of the vesicles contained in individual nerves, it is likely that the VIP-immunoreactive nerve fibers in the medulla and the juxtamedullary cortex are derived from the medullary VIP-ganglion cells, while those in the superficial cortex are of extrinsic origin. The immunoreactive nerve fibers in both the cortex and the medulla were often in direct contact with cortical cells and chromaffin cells, where no membrane specializations were formed. The immunoreactive nerve fibers were sometimes associated with the smooth muscle cells and pericytes of small blood vessels in the superficial cortex. In addition they were often seen in close apposition to the fenestrated endothelial cells in the cortex and the medulla, only a common basal lamina intervening. Several possible mechanisms by which VIP may exert its effect in the adrenal gland are discussed.     

c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine

 c-kit immunohistochemistry was performed on unfixed frozen sections of human small (duodenum, jejunum, and ileum) and large intestine (ascending, transverse, descending, and sigmoid colon). The c-kit immunoreactive cells in the muscularis externa of the intestinal wall were identified as interstitial cells of Cajal (ICC) and mast cells. ICC were identified by their morphology, localization, and organization based on previous light and electron microscopic studies. In the small intestine, ICC were located primarily in relation to the myenteric plexus of Auerbach, but also in septa between circular muscle lamellae. In the large intestine, ICC were seen in relation to Auerbach’s plexus, but also and in great numbers in the circular muscle layer and in teniae of the longitudinal muscle layer. The morphology of the ICC was similar in the small and large intestine, but the pattern of distribution was obviously different. c-kit immunoreactive mast cells were found predominantly in the inner part of the circular muscle layer. The anti-c-kit method is found to be an easy and reliable method to study at least most of the interstitial cells of Cajal and thereby contribute to further normal and pathological studies. Accepted: 31 July 1997     

Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion

The origin of nitric oxide synthase-containing nerve fibers in rat celiac-superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the cceliac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons.     

An histological and immunocytochemical study of the neuroendocrine cells in the intestine of Podarcis hispanica Steindachner, 1870 (Lacertidae)

Summary Numerous endocrine cells can be observed in the gut of the lizard Podarcis hispanica after application of the Grimelius silver nitrate technique. The argyrophilic endocrine cells are usually tall and thin in the small intestine but short, basal, and round in the large intestine. Eleven types of immunoreactive endocrine cells have been identified by immunocytochemical methods. Numerous serotonin-, caerulein/gastrin/cholecystokinin octapeptide-and peptide tyrosine-tyrosine-immunoreactive cells; a moderate number of pancreatic polypeptide-, neurotensin-, somatostatin-, glucagon-like peptide-1-and glucagon-immunoreactive cells, and few cholecystokinin N-terminal-and bombesin-immunoreactive cells were found in the epithelium of the small intestine. Coexistence of glucagon with GLP-1 or PP/PYY has been observed in some cells. In the large intestine a small number of serotonin-, peptide tyrosine-tyrosine-, pancreatic polypeptide-, neurotensin-, somatostatin-and glucagon-like peptide-1-immunoreactive cells were detected. Vasoactive intestinal peptide immunoreactivity was found in nerve fibers of the muscular layer. Substance P-immunoreactive nerve fibers were detected in lamina propria, submucosa and muscular layer. Chromogranin A-immunoreactive cells were observed throughout the intestine, although in lower numbers than argyrophilic cells.     

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine. Received: 10 April 1996 / Accepted: 13 May 1996     

VIP like immunoreactive nerves in human respiratory tract. Light and electron microscopic study

The present study provides light and electron microscopical evidence of Vasoactive Intestinal Peptide - (VIP) like immunoreactive nerves in human lower respiratory tract. Peroxidase antiperoxidase (PAP) technique was used to localize VIP-like immunoreactivity light microscopically and ultrastructurally. Under light microscopy, VIP-like immunoreactive nerves were observed in the smooth muscle layer of secondary bronchi to small bronchioli, and in bronchial glands. In addition, positive immunoreactive nervous network to VIP was found around nerve cell bodies in small microganglia. The bronchial epithelium of airway tract did not receive any VIP positive nerve fibers. Ultrastructurally VIP-like positive immunoreaction was localized in large granular vesicles ranging from 90 to 210 nm. Usually VIP-like positive immunoreactive nerve profiles contained several immunoreactive large vesicles (100-210). However, nerve profiles containing only a few positive large vesicles (80-150) were also observed. Under electron microscopy VIP-positive nerve profiles corresponded ultrastructurally to nerve profiles containing large granular vesicles observed in conventional electronmicroscopy. The present study provides new information about the innervation of human lower airway tract and widens the concept of their functional regulation on the anatomical basis reported here.     

Ultrastructural analysis of spinal primary afferent fibers within the circular muscle of the cat lower esophageal sphincter

We have analyzed the ultrastructural characteristics and environment of spinal primary afferent fibers that run within the circular muscle of the cat lower esophageal sphincter. These were selectively labeled by anterogradely transported cholera toxin B subunit conjugated with horseradish peroxidase. Most of the labeled fibers were perpendicular to the muscle cells but some ran sinuously or parallel to the muscle cells. All the labeled fibers were unmyelinated and exhibited relatively rare varicosities. Most of the fibers were in large nerve fiber bundles surrounded by perineurium and probably project to the mucosa. Only some fibers that were in small nerve fiber bundles with no perineurium ran parallel to the musculature and established close relationships with smooth muscle cells. They might be a small subpopulation of the spinal tension receptors, most of the other spinal tension receptors being located in the myenteric plexus area, between the circular and longitudinal muscle. Accepted: 2 December 1999     

Effects of met-enkephalin on the mechanical activity and distribution of met-enkephalin-like immunoreactivity in the cat small intestine

Naloxone-dependent effects of Met-enkephalin (10(-8) M) on the spontaneous and electrically induced mechanical activities were studied in longitudinal and circular preparations isolated from the cat duodenum, jejunum and ileum. Met-Enkephalin changed the spontaneous activity of all preparations tested with the exception of the circular preparations from the ileum. Met-Enkephalin-induced responses of the longitudinal preparations from the ileum were abolished by treatment with tetrodotoxin (10(-7) M), while the responses of both longitudinal and circular preparations from the duodenum and jejunum were only partially depressed, being resistant to tetrodotoxin components. The latter were most pronounced in the duodenum. The neurogenic electrically induced (0.5 msec, 5 Hz, 150 pulses) responses of all the preparations consisted mainly of contractile components which were significantly and naloxone-dependently reduced by Met-enkephalin (10(-8) M). The contractile components of the responses, which were reduced by Met-enkephalin, were entirely abolished by atropine (3 x 10(-6) M). Both Met-enkephalin and atropine inhibitory effects on the neurogenic responses were more pronounced in the ileum. Met-Enkephalin was found in nerve fibers of the myenteric plexus distributed mainly among the circular muscle. Single immunoreactive nerve fibers were observed in the longitudinal muscle layer of the duodenum but not in the jejunum and ileum. The distribution of Met-enkephalin-like immunoreactivity along the small intestine did not show significant differences among the three intestinal regions tested. The results obtained suggest that Met-enkephalin can modulate the mechanical activity of the cat small intestine, inhibiting cholinergic transmission and/or activating smooth muscle opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum)

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent . After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.     

Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 7m. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.     

Characterization of immunoreactive motilin from the rat small intestine.

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.