Substance P-containing nerves in the human small intestine. Distribution, ultrastructure, and characterization of the immunoreactive peptide

Light and electron microscopic immunocytochemical techniques were used to examine the distribution and ultrastructure of substance P-immunoreactive nerves in human jejunum and distal ileum. The organization of human enteric substance P-containing nerves closely resembled that in other species. Dense arrays of varicose immunofluorescent fibers occurred in myenteric and submucous ganglia (which contained immunoreactive nerve cell bodies) and in the mucosa. There were fibers in both muscle layers, in the muscularis mucosae, and around blood vessels. Fibers in the myenteric plexus contributed to both ascending and descending pathways. Substance P-immunoreactive axon profiles contained small round and large round vesicles and were apposed to nerve cell bodies, and nonimmunoreactive and immunoreactive axon profiles. Synapselike contacts were occasionally observed on nerve cell bodies and processes. The substance P-like material was characterized by high pressure liquid chromatography and radioimmunoassay and found to be indistinguishable from the authentic undecapeptide. These results suggest that enteric nerves containing substance P may play similar roles in humans as in other species.     

Origins of peptide and norepinephrine nerves in the mucosa of the guinea pig small intestine

Norepinephrine, acetylcholine, and certain peptides are contained in mucosal nerves and have potent effects on transepithelial water and electrolyte fluxes. It is difficult to ascribe roles for these nerves as their sources are unknown. The present studies were undertaken to determine the origins of nerve fibers that are found in the mucosa of the guinea pig small intestine and which contain one of the following substances: vasoactive intestinal peptide, substance P, somatostatin, neuropeptide Y, cholecystokinin, or norepinephrine. Nerve fiber origins were ascertained by making lesions to sever pathways through which the nerves could reach the mucosa. The lesioning operations were homotopic autotransplants of short (2 cm) segments of intestine; myectomies, in which a 5-10-mm length of intestine was stripped of longitudinal muscle and myenteric plexus; and extrinsic denervation, in which nerves reaching the intestine through the mesentery were severed. The results of these studies, considered along with previously published work, led to the upcoming conclusions. Nerve fibers in the mucosa showing immunoreactivity for vasoactive intestinal peptide, somatostatin, cholecystokinin, and neuropeptide Y arise from cell bodies in the overlying submucous plexus. Substance P fibers arise in part from the overlying submucous plexus and in part from the overlying myenteric plexus. Mucosal norepinephrine fibers arise from extrinsic sympathetic ganglia. Enkephalin, gastrin-releasing peptide, and 5-hydroxytryptamine, which are in some enteric nerves, are not found in submucous nerve cells and few, if any, fibers containing these substances supply the mucosa. Thus, the mucosa receives a dense nerve supply, much of which arises locally from submucous ganglia.     

Peptide-containing nerve fibers in the stomach wall of rat and mouse

Peptide-containing nerve fibers were found to be numerous in the glandular stomach of the rat and mouse. The immunoreactive neuropeptides demonstrated included vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP), substance P (SP), enkephalin, somatostatin, cholecystokinin, and neuropeptide Y (NPY). The density and distribution of the various peptide-containing fibers did not differ overtly between the pyloric and oxyntic gland areas except for the GRP fibers, which were fewer in the pyloric than in the oxyntic mucosa. The entire VIP nerve fiber population was found to also contain PHI. Immunoreactive NPY was found to occur in the VIP/PHI fibers (VIP/PHI/NPY fibers) in the smooth muscle and intramural ganglia of both rat and mouse and in the mucosa of the mouse. Mucosal VIP/PHI fibers in the rat did not contain any NPY-like material. Perivascular NPY fibers in both species and mucosal NPY fibers in the rat did not contain VIP or PHI. The mucosa harbored numerous GRP fibers and VIP/PHI (rat) or VIP/PHI/NPY (mouse) fibers, and a modest number of NPY (rat) and SP fibers. In the submucosa the peptide-containing nerve fibers were found mainly in the ganglia and around blood vessels. Blood vessels received a rich supply of NPY fibers; the number of perivascular VIP/PHI, GRP, and SP fibers was much lower by comparison. The smooth muscle and myenteric ganglia harbored not only VIP/PHI/NPY, GRP, and SP fibers but also enkephalin, somatostatin, and cholecystokinin fibers. Gastrin-releasing peptide, VIP/PHI/NPY, SP, and enkephalin nerve cell bodies occurred in the myenteric ganglia. As studied in the rat, vagal denervation did not affect the density and distribution of the various peptide-containing nerve fibers. After sympathectomy, mucosal and perivascular NPY fibers disappeared. The other types of peptide-containing nerve fibers were not affected.     

Distributions of neuropeptides in the human esophagus

The distributions of nerve cells and fibers with immunoreactivity for the peptides substance P, somatostatin, enkephalin, vasoactive intestinal peptide, gastrin-releasing peptide, and neuropeptide Y and the enzyme tyrosine hydroxylase were examined in 25 samples of human esophagus. These were compared with samples of stomach and intestine. In the smooth muscle of the muscularis externa, the muscularis mucosae, and beneath the epithelium, the most abundant nerve fibers contained vasoactive intestinal peptide and neuropeptide Y, in contrast to the scarcity of substance P, enkephalin, somatostatin, and gastrin-releasing peptide. Gastric and intestinal samples contained dense populations of fibers containing vasoactive intestinal peptide, neuropeptide Y, substance P, and enkephalin in the equivalent layers, but somatostatin- and gastrin-releasing peptide-immunoreactive fibers were scarce. Complete coexistence of vasoactive intestinal peptide and neuropeptide Y in nerve fibers within the muscle layers was demonstrated in the esophagus, but not in gastric and intestinal samples. The myenteric plexus along the length of the esophagus contained cell bodies and fibers reactive for vasoactive intestinal peptide, neuropeptide Y, enkephalin, and substance P. Somatostatin-immunoreactive cell bodies were very rare in the myenteric plexus, no gastrin-releasing peptide-immunoreactive cell bodies were seen, and both somatostatin and gastrin-releasing peptide-immunoreactive fibers were rare. In the upper esophagus, striated muscle bundles did not contain nerve fibers reactive for these peptides but immunoreactive fibers were seen in the muscularis mucosae and subepithelium. It is concluded that the esophagus has a different pattern of innervation by peptide-containing neurons than the stomach and intestines. Esophageal neurons can be classified into separate classes on the basis of their peptide content.     

Vasoactive intestinal polypeptide levels in sigmoid colon in idiopathic constipation and diverticular disease.

The distribution in the bowel wall of vasoactive intestinal polypeptide-, neuropeptide Y-, and substance P-containing nerve cell bodies and nerve fibers has been described in human sigmoid colon by immunohistochemical examination. In patients with chronic idiopathic constipation, diverticular disease, and in controls (of tissue taken from patients with carcinoma, from a site distant from the tumor that appeared macroscopically normal), the concentrations of vasoactive intestinal polypeptide, neuropeptide Y, and substance P have been measured by immunoassay in the following preparations of sigmoid colon: mucosa, whole colonic wall with mucosa dissected away, circular muscle, and taenia coli. In idiopathic constipation, the vasoactive intestinal polypeptide content of the whole wall minus mucosa was reduced when compared with controls (P less than 0.05) but was unaltered in the mucosa, circular muscle, and taenia coli. In diverticular disease, the vasoactive intestinal polypeptide content of the mucosa and whole wall minus the mucosal layer was increased when compared with control tissue (P less than 0.05 and P less than 0.02, respectively) but was unaltered in the circular muscle and taenia coli. Substance P and neuropeptide Y levels in all layers of colonic wall were unaltered in these two diseases. The disturbances in the normal neural content of vasoactive intestinal polypeptide in the bowel wall in idiopathic constipation and diverticular disease may initiate or contribute to the functional changes seen in these disorders.     

Selective damage of intrinsic calcitonin gene-related peptide-like immunoreactive enteric nerve fibers in streptozotocin-induced diabetic rats

The distribution of calcitonin gene-related peptidelike immunoreactive (CGRP-LI) nerve fibers in the myenteric plexus of ileum and proximal colon of rats 8 wk after induction of diabetes with streptozotocin was studied using immunohistochemical techniques. A marked decrease in CGRP-LI nerve fibers mainly around the ganglion cells of the myenteric plexus of both ileum and proximal colon was observed in diabetic rats. The sparsely located immunoreactive nerve cell bodies in the control rats were absent in the diabetic preparations. There were, however, intensely stained CGRP-LI varicose nerve fibers that ran through the internodal strands and over the myenteric ganglia of the diabetic intestines. These findings indicate the presence of CGRP-LI nerve fibers of dual origin in the intestinal wall. The absence of positive cell bodies and diminished CGRP-LI nerve fibers around the ganglion cells in the diabetic tissues suggest that the state of diabetes selectively affects CGRP-LI nerve fibers of intrinsic rather than extrinsic origin. Furthermore, the absence of change in substance P-like immunoreactivity in the enteric system of rats with streptozotocin-induced diabetes of the same duration suggests that calcitonin gene-related peptide and substance P are contained in different populations of intrinsic nerve fibers in the gastrointestinal tract of the rat.     

Electron microscopic study of neuropeptide Y-containing nerve elements of the guinea pig small intestine

Neuropeptide Y-containing nerve cell bodies and processes were identified by electron microscopic immunocytochemistry in the guinea pig small intestine. Labeled nerve processes were numerous in the myenteric plexus. However, a few immunoreactive nerve fibers were found in all layers of the small intestine. Some of the immunoreactive nerve processes were found in close apposition to the epithelial cells of the crypts of Lieberkühn and to endothelial and smooth muscle cells. The neuropeptide Y-containing nerve cell bodies were preferentially located in the submucous ganglia. In the myenteric plexus many synaptic connections were observed between the neuropeptide Y-immunoreactive nerve fibers and unlabeled nerve cell bodies and other nerve fibers. These findings provide a morphologic basis for the possibility that neuropeptide Y may act as a transmitter and exert postsynaptic effects on intrinsic neurons, in addition to participating in the regulation of smooth muscle activity and epithelial cell functions.     

Abnormalities of peptide-containing nerve fibers in infantile hypertrophic pyloric stenosis

The distributions of nerve cells and fibers with immunoreactivity for the peptides enkephalin, gastrin-releasing peptide, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide were examined in specimens of myenteric plexus and external muscle from the pylorus of 20 infants with hypertrophic pyloric stenosis. These were compared with peptide distributions in pyloric samples from unaffected infants and adults. In the normal pylorus the circular muscle was richly supplied with fibers reactive for enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. In pyloric stenosis, these immunoreactive fiber bundles were either missing or less than 5% of normal. In contrast, there were reactive cell bodies and nerve fibers in the myenteric plexuses of both normal and affected specimens. In the samples from cases of stenosis, swollen nerve fibers that appeared to be in the process of degeneration were frequently encountered. It is concluded that infantile hypertrophic pyloric stenosis is associated with a loss of peptide immunoreactivity in nerve fibers in the circular muscle, although the same peptides are still revealed in fibers and in nerve cell bodies in the myenteric plexus.     

Serotoninergic neurons in human fetal intestine: an immunohistochemical study

The distribution of 5-hydroxytryptamine-like immunoreactivity was studied in whole-mount preparations of intestine from human fetuses. Immunoreactive nerve cell bodies were located in the myenteric plexus and were occasionally found in the submucous plexus; they were often seen to have long processes. Varicose fibers were found in the ganglia and internodal strands of the myenteric and submucous plexuses, in the deep muscular plexus of the circular muscle, and in the walls of some small mesenteric blood vessels immediately outside the intestine. This study provides evidence for the presence of serotoninergic nerves in the human intestine.     

Neuropeptides in the thyroid gland: distribution of substance P and gastrin/cholecystokinin and their effects on the secretion of iodothyronine and calcitonin

Previously, vasoactive intestinal polypeptide was localized to intrathyroidal nerve fibers. It stimulates iodothyronine secretion in mice. In the present study two populations of nerve fibers containing substance P and gastrin/cholecystokinin (CCK)-like immunoreactivity, respectively, were demonstrated in the thyroid gland of several mammals. The substance P fibers occurred around blood vessels and follicles, whereas the gastrin/CCK fibers occurred mainly around follicles. In the chicken thyroid and ultimobranchial glands only substance P-containing fibers could be demonstrated. Such fibers were particularly numerous in the ultimobranchial gland. CCK-4, CCK-8, and substance P did not increase thyronine secretion measured as release of radioiodine into the circulation of mice pretreated with Na125I and T4. The TSH-induced release of radioiodine was also unaffected. Calcitonin secretion in rats was stimulated by CCK-4, CCK-8, substance P, and vasoactive intestinal polypeptide.     

Enteric nerves in diabetic rats: increase in vasoactive intestinal polypeptide but not substance P

The distribution of vasoactive intestinal polypeptide (VIP) and substance P-like immunoreactivities was studied by immunohistochemistry in the myenteric plexus and circular muscle layer of the ileum and proximal colon of rats 8 wk after induction of diabetes with streptozotocin. A consistent increase was observed in fluorescence intensity of VIP-like immunoreactivity in the nerve fibers, and intensely stained cell bodies were significantly more frequent in the myenteric plexus of the ileum (p less than 0.001) from diabetic animals. Some varicosities of VIP-like immunoreactive fibers in the myenteric plexus appeared to be enlarged. Vasoactive intestinal polypeptide-like immunoreactivity was increased and VIP-like immunoreactive nerves appeared thicker in the circular muscle layer of both diabetic ileum and proximal colon. The VIP levels were measured biochemically in tissue consisting of the smooth muscle layers and myenteric plexus. A significant increase in the VIP content per centimeter of intestine was found in both the ileum (p less than and proximal colon (p less than 0.01) from diabetic rats. In contrast, no apparent change in substance P innervation was observed immunohistochemically in the myenteric plexus and circular muscle layer of either diabetic ileum or proximal colon when compared with controls. The results are discussed in relation to the symptoms of autonomic neuropathy of the gut in diabetes.     

Migration of the myoelectric complex after interruption of the myenteric plexus: intestinal transection and regeneration of enteric nerves in the guinea pig

The effects of surgical interruption of the myenteric plexus (myectomy), extrinsic denervation of a length of small intestine, or transection and reanastomosis of the intestinal wall on migration of phase III of the migrating myoelectric complex was studied in guinea pigs. In addition, the recovery of phase III migration and the regrowth of intestinal nerves and muscle across the reanastomosis was studied at various times up to 60 days after surgery. At 6-9 days after surgery, phase III did not migrate past the myectomy during 50%-60% of recorded migrating myoelectric complexes and transection and reanastomosis of the intestinal wall blocked aboral progression of phase III in 90% of cases. Extrinsic denervation did not alter phase III migration through the denervated segment. Phase III migration past the reanastomosis recovered with time after surgery; 80% recovery occurred by 60 days after surgery. Immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin were used as markers for intestinal nerves that were cut by transaction. Immunoreactivities for vasoactive intestinal peptide and gastrin-releasing peptide are contained in myenteric neurons that project in an oral to anal direction to other myenteric ganglia and to the circular muscle. Immunoreactivity for somatostatin is contained in nerve fibers projecting aborally to other myenteric ganglia. At 7-15 days after surgery, there were accumulations of immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin in nerve fibers on the oral side of the reanastomosis, but nerve fibers containing these peptides were not observed in myenteric ganglia or circular muscle close to the anal edge. At 23-28 days, immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin nerve fibers were traced across the reanastomosis and nerve terminals were detected in ganglia and muscle close to the lesion on the anal side. Nerve fibers traversed the lesion in all cases at 57-60 days and vasoactive intestinal peptide-, gastrin-releasing peptide-, and somatostatin-immunoreactive nerve terminals were detected in the first two to three rows of myenteric ganglia on the anal side. Regrowth of intestinal muscle followed a similar time-course to that observed for nerves. These data suggest that interruption of the myenteric plexus alone does not completely block phase III migration. In addition, recovery of phase III migration past a reanastomosis is associated with a restoration of both nervous and mechanical connections.     

Effects of substance P and vasoactive intestinal polypeptide on contractile activity and epithelial transport in the ferret jejunum.

Previous studies in the ferret demonstrated that vagal nerve stimulation induced an atropine-resistant water secretion. Substance P and vasoactive intestinal polypeptide are possible mediators of this secretory response. The objectives of this study were to investigate the in vivo effects of substance P and vasoactive intestinal polypeptide on the jejunal musculature and epithelium. Substance P caused an increase in jejunal motility, water secretion, and transmural potential difference. Cholinergic blockade did not affect the substance P-induced contractions, but did reduce the increase in transmural potential difference, suggesting an inhibition of water secretion. Vasoactive intestinal polypeptide abolished motor activity; however, it induced an increase in transmural potential difference that was atropine and tetrodotoxin resistant. By immunohistochemical methods, immunoreactive vasoactive intestinal polypeptide and immunoreactive substance P were localized to both nerve cell bodies and nerve fibers in the ferret intestine. Determination of intestinal concentrations of vasoactive intestinal polypeptide and substance P in the ferret showed concentrations of these two neuropeptides that were similar to those in human intestine and demonstrated much higher concentrations of these substances in the muscular layer than in the epithelial layer. Our data demonstrate that in the ferret substance P excites and vasoactive intestinal polypeptide inhibits jejunal motor activity. However, both peptides increase water secretion. Our results suggest that in response to vagal stimulation, neuronally released substance P or vasoactive intestinal polypeptide may participate in the atropine-resistant water secretion.     

Substance P modulation of acetylcholine-induced currents in embryonic chicken sympathetic and ciliary ganglion neurons.

Substance P has been identified by combined immunohistochemical and radioimmunological techniques to be present in preganglionic cholinergic and sensory nerve fibers of amphibian, mammalian, and avian autonomic ganglia. The peptide has been shown to depolarize sympathetic neurons of frog and guinea pig and to decrease the cholinergic activation of Na+ influx and catecholamine release from chromaffin cells. The aim of this study was to examine the interaction of acetylcholine and substance P on autonomic neurons. This report demonstrates a direct effect of substance P on acetylcholine-induced inward currents in both sympathetic and parasympathetic neurons clamped near resting membrane potential. Under these conditions, substance P dramatically enhances the rate of decay of the inward current in the continued presence of agonist without substantially affecting peak inward current. This effect is consistent with an enhancement of acetylcholine-receptor desensitization. Since substance P-containing cell bodies have been demonstrated in the avian (preganglionic) column of Terni as well as in fibers from the nucleus of Edinger-Westphal, the observed peptide inhibition of cholinergic activation of the neurons may function physiologically to modulate synaptic function in autonomic ganglia.     

Distribution and origin of the peripheral innervation of rat cervical esophagus

Several neurotransmitters, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), galanin, enkephalin, calcitonin-gene related peptide (GGRP), substance P, as well as nitric oxide synthase (NOS), and the noradrenergic marker tyrosine-hydroxylase (TH) were localized by immunocytochemistry in the cervical esophagus of rat. Nerve fibers containing the neuropeptides, NOS, and TH were distributed in the myenteric plexus, around muscle bundles and small blood vessels. Injection of the retrograde tracer True Blue (TB) into the cervical esophagus resulted in the appearance of labeled nerve cell bodies in the superior cervical, the stellate, the nodose, the sphenopalatine, the dorsal root ganglia at levels C₂–C₇, and in local ganglia close to the thyroid. Most of the TB-labeled nerve cell bodies in the superior cervical ganglia contained NPY. In the stellate ganglion, a few labeled nerve cell bodies contained VIP whereas an additional few cell bodies stored NPY. In local ganglia, the majority of labeled cell bodies contained VIP. In the nodose ganglion and cervical dorsal root ganglia, the majority of the labeled nerve cell bodies stored CGRP. The results indicate that the cervical esophagus has a dense innervation with multiple neurotransmitters emanating from several ganglia. As judged by the pattern of nerve fiber distribution, they may regulate esophageal peristalsis and blood flow, some of them possibly in a cooperative manner.     

Morphological study on the motility of the human colon

We tried to make a clear three-dimensional picture of the autonomic nerves in the wall of the human colon, using a Golgi method rarely applied to human materials. At autopsy, sigmoid colon without mucosal lesions were collected from 16 males after sudden death from apoplexy, head injury, or myocardial infarction. These materials were fixed in 10% formalin, impregnated with a modified Golgi method and embedded in celloidin. Then three-dimensional serial sections were made and observed with a light microscope. Many fine nerve fibers formed a plexus in the subserosa, muscular layer, submucosa, and mucosa. The myenteric plexus was made up of rectangular meshes of nerve fiber bundles. However, unlike myenteric plexus, no regular mesh was found in the submucosal plexus. Further, nerve fibers connecting myenteric and submucosal plexus were observed. It may be concluded from these findings that there exist nerve pathways regulating intestinal motility between myenteric and submucosal plexus.     

Expression of cholecystokinin A receptors in neurons innervating the rat stomach and intestine.

BACKGROUND & AIMS: Two distinct receptors, cholecystokinin (CCK)-A and CCK-B, mediate CCK effects in the digestive system. The aim of this study was to elucidate the cellular sites of expression of CCK-A receptor in the rat stomach and small intestine. METHODS: We developed and characterized an antibody to the N-terminal region (LDQPQPSKEWQSA) of rat CCK-A receptor and used it for localization studies with immunohistochemistry. RESULTS: Specificity of the antiserum was demonstrated by (1) detection of a broad band at 85-95 kilodaltons in Western blots of membranes from CCK-A receptor CHO-transfected cells; (2) cell surface staining of CCK-A receptor-transfected cells, (3) translocation of CCK-A receptor immunostaining in CCK-A receptor-transfected cells after exposure to CCK; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with the peptide used for immunization. CCK-A receptor immunoreactivity was localized to myenteric neurons and to fibers in the muscle and mucosa. In the stomach, myenteric neurons and mucosal fibers were abundant. Many CCK-A receptor myenteric neurons contained the inhibitory transmitter vasoactive intestinal polypeptide, and some were immunoreactive for the excitatory transmitter substance P. Subdiaphragmatic vagotomy reduced the density of CCK-A receptor fibers in the gastric mucosa by approximately 50%, whereas celiac/superior mesenteric ganglionectomy had no detectable effect on fiber density. CONCLUSIONS: CCK-A receptor is expressed in functionally distinct neurons of the gastrointestinal tract. CCK-A receptor may mediate reflexes stimulated by CCK through the release of other transmitters from neurons bearing the receptor.     

Peptide-containing nerve fibres in the gut wall in Crohn's disease.

Neurones containing VIP, substance P, or enkephalin were studied by immunocytochemistry in intestinal specimens from 27 patients with Crohn's disease. Also several endocrine cell systems in the gut were examined. The results were compared with those from a control group of 26 patients. The relative frequency of various endocrine cells did not differ overtly from that in controls. Vasoactive intestinal polypeptide and substance P nerve fibres were distributed in all layers of the gut wall, including the submucosal and myenteric plexuses, whereas enkephalin fibres were restricted to the smooth muscle layer and the myenteric plexus. The distribution and frequency of the peptide-containing nerve fibres were the same in Crohn's disease patients as in control patients. A proportion of these nerve fibres, however, were notably coarse in the Crohn's disease patients. This was particularly apparent in the afflicted parts of the intestine although it was noted also in non-afflicted parts. The concentration of VIP and substance P (expressed as pmol/g wet weight) did not, however, exceed that of the control group.     

Slow transit chronic constipation (Arbuthnot Lane's disease)

Seven patients (6 women, 1 man) with severe idiopathic chronic constipation, who underwent surgery with subtotal colectomy and ileorectal anastomosis, were investigated for the occurrence and density of nerve fibres, immunoreactive to different neuropeptides in the mucosa, submucosa, ganglia and smooth muscle in fresh specimens from the colon ascendens, the colon transversum and the colon descendens-sigmoideum. The following substances were studied: enkephalin, substance P, somatostatin, neuropeptide Y, vasoactive intestinal polypeptide, calcitonin gene-related peptide, bombesin, motilin, tyrosine hydroxylase, dynorphin and galanin. Nerve fibres immunoreactive to CGRP occurred in large numbers in the myenteric ganglia of the patients with severe idiopathic chronic constipation, whereas in the myenteric ganglia of the control cases they only occurred in low numbers. In two patients there was no detectable motilin immunoreactivity and in one patient only sparse in the mucosa and the smooth muscle. The other neuropeptides investigated occurred in the density and distribution previously reported in the normal gut. With the present technique there were indications that patients with severe idiopathic chronic constipation have a significant difference in the occurrence of immunoreactive nerve fibres to CGRP and motilin compared to control patients.     

Distribution and Colocalization of Nitric Oxide Synthase and Calretinin in Myenteric Neurons of Developing, Aging, and Crohn's Disease Human Small Intestine

The pattern of distribution and colocalizationof nitric oxide synthase and the calcium-binding proteincalretinin in myenteric neurons and nerve fibers wereexamined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normaladults (mean age 50 years old), old age (mean age 80years old), and Crohn's disease patients (mean age 30years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all agegroups investigated, NADPH-diaphorase-reactive andcalretinin-immunoreactive neurons and nerve fibers wereseen throughout the myenteric plexus. The highestproportion of NADPH-diaphorase-reactive neurons was foundin the myenteric ganglia of old age intestines (56% ofprotein gene product-immunoreactive neurons) followed byfetal intestines (41%) and Crohn's intestine (30%) compared with intestines of controladults (20%). A similar trend was observed forcalretinin-immunoreactive neurons where the highestproportion of immunoreactive neurons was found in themyenteric ganglia of old age intestines (28% of proteingene product-immunoreactive neurons), followed by fetalintestines (22%), and Crohn's intestines (18%) comparedwith intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretininimmunoreactivity was only seen in the myenteric neuronsof fetal intestines (2% of NADPH-diaphorase-reactiveneurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive andcalretinin-immunoreactive neurons in the myentericganglia of fetal intestine differs from that of theother age groups. In the fetal intestine, the myenteric neurons containing either calretinin orNADPH-diaphorase are distributed through out themyenteric ganglia with no specific orientation to oneanother. In the intestines of control adult, Crohn's,and old age patients, single largecalretinin-immunoreactive neurons are surrounded by anumber of small NADPH-diaphorase-positive neurons, withthis feature being more prominent in intestines ofold-age and Crohn's disease patients. In summary, a high number ofboth NADPH-diaphorase-reactive andcalretinin-immunoreactive neurons were seen in themyenteric ganglia of fetal, old age, and Crohn'sintestines; we discuss that there may be a role for nitric oxide andcalretinin in the process of development, aging, andpathological changes in the human intestine associatedwith alteration in the calcium homeostasis in the myenteric neurons.