Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the gastrointestinal tract of rat and pig

One of the opioid precursor molecules, pre-pro-enkephalin A, contains within it, in addition to Leu-enkephalin (Leu-Enk) and Met-enkephalin (Met-Enk), Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-8), which is specific to this precursor. This study deals with the localization of Met-Enk-8-like immunoreactivity in the gastrointestinal tract of rat and pig. Immunoreactivity was identified in intramural nerve elements of rat and pig, and in gut endocrine cells of pig. Immunoreactive (IR) nerve fibers were seen mainly in the myenteric plexus of rat and in both the myenteric and submucosal plexuses of pig. Some IR fibers were dispersed throughout the lamina propria mucosae of rat. Porcine IR endocrine cells were dispersed in the epithelium from the pyloric antrum to the ileum, existing concomitantly with enterochromaffin (EC) cells. Specificity tests revealed that immunoreactivity to Met-Enk-8 antiserum was not influenced by preincubation of the antiserum with Leu-Enk and Met-Enk. This suggests the possibility that pre-pro-enkephalin A is contained in the gastroenteric nerves of rat and pig and in a population of porcine EC cells.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Dynorphin-A (1-8) is contained within perikarya, nerve fibres and nerve terminals of rat duodenum

By the use of well-characterized antibodies against porcine dynorphin-A(1-8), an endogenous opioid peptide, and the use of a modified immunofluorescence microscopic technique, dynorphin-A(1-8) stained perikarya, nerve fibres, and nerve terminals were visualized in the rat duodenum. Dynorphin-A(1-8) immunoreactive perikarya were revealed with certainty only in the myenteric plexus, while dynorphinergic nerve fibres could bee seen in the myenteric plexus and circular muscle layer, but not in the longitudinal muscle layer and submucous plexus. Dynorphin-A(1-8) immunofluorescent nerve endings were in close contacts with submucosal blood vessels, probably arterioles, and Brunner's gland cells. These findings suggest that the opioid peptide dynorphin-A(1-8) might be synthetized within myenteric plexus perikarya of the rat duodenum and that it might modulate the peristaltic activity, intestinal blood pressure, and production of mucopeptides synthetized within Brunner's gland cells.     

Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species

Summary The distribution of galanin-immunoreactive (GAL-IR) neurons was mapped in detail in the gastro-intestinal tract of the rat, mouse, guinea-pig and pig by use of the indirect immunofluorescence technique. GAL-IR cell bodies were found in both the submucous and the myenteric plexus, with considerably higher numbers in the former ganglia. The largest number of GAL-IR perikarya was seen in the duodenal submucous plexus of the pig. With some (single) exceptions, GAL-IR cell somata were not observed in the myenteric plexus of the pig and guinea-pig, and in the submucous plexus of the esophagus and the stomach of the guinea-pig.GAL-IR fibers ocurred in most parts of the gastro-intestinal tract. In the lamina propria a few non-varicose, weakly fluorescent fibers were noted in the mouse and rat, whereas in the pig and guinea-pig were large numbers of GAL-IR fibers with a varicose appearance was observed. These fibers were in all species most numerous in the distal portion of the intestinal tract. In the submucosa GAL-IR fibers were detected in all four species, and in the pig and guinea-pig some fibers surrounded blood vessels. A large number of GAL-IR fibers was generally seen in the circular smooth muscle layer, except in the guinea-pig, which only seemed to contain a few fibers. In the longitudinal muscle layer only single fibers could be detected. However, the gastric fundus region of the pig contained a moderate number of fibers in the longitudinally and obliquely oriented layers.In general, in the rat, mouse and pig, the submucous and myenteric plexus contained moderate or large numbers of GAL-IR fibers. In the guinea-pig, no or only single fibers were observed in the plexus of the upper gastro-intestinal tract and the rectum, while moderate numbers were seen in the ileum and colon.Thin adjacent sections stained for vasoactive intestinal polypeptide (VIP) and GAL revealed the coexistence of these two peptides in cell bodies of the myenteric plexus in the pig duodenum and guinea-pig colon. In these two species the GALand VIP-nerve fiber networks also exhibited marked similarities. However, in the rat and mouse VIPand GAL-distribution patterns were in general different.The present findings indicate the presence of yet another neuropeptide or peptide family in the gastro-intestinal tract of several rodents and the pig.     

Histamine-containing peripheral neuronal and endocrine systems

An immunohistochemical method was developed to detect histamine in tissues. The aim of this study was to reveal the cellular stores of histamine in the gastrointestinal tract, pituitary, and adrenal gland. Histamine-containing nerve fibers were found in both rat and guinea pig gut. The origin of at least some of these fibers in the rat ileum was the submucous ganglion cell layer. In the rat stomach, numerous enterochromaffin-like cells exhibited histamine immunofluorescence, and endocrine cells in the ileum and jejunum contained histamine. Only mast cells contained histamine in the neurohypophysis. A large number of process-bearing cells in the guinea pig but not in the rat adrenal medulla contained histamine. The study shows that histamine is present in peripheral nerves and endocrine cells in addition to mast cells, and may function as a neurotransmitter or hormone.     

Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig

Summary Neuromedin U immunoreactivity was located histochemically in the guinea-pig small intestine. Projections of immunoreactive neurons were determined by analysing patterns of degeneration following nerve lesions. The co-localization of neuromedin U immunoreactivity with immunoreactivity for substance P, neuropeptide Y, vasoactive intestinal peptide and calbindin was also investigated. Neuromedin U immunoreactivity was found in nerve cells in the myenteric and submucous plexuses and in nerve fibres in these ganglionated plexuses, around submucous arterioles and in the mucosa. Reactive fibres did not supply the muscle layers. Most reactive nerve cells in the myenteric ganglia had Dogiel type-II morphology and in many there was co-localization of calbindin, although some Dogiel type-II neuromedin U neurons were calbindin negative. Lesion studies suggest that these myenteric neurons project circumferentially to local myenteric ganglia. Projections from myenteric neurons also run anally in the myenteric plexus, while other projections extend to submucous ganglia, and still further projections run from the intestine to provide terminals in the coeliac ganglia. In the submucous ganglia neuromedin U was co-localized in three populations of nerve cells: (i) those with vasoactive intestinal peptide immunoreactivity, (ii) neurons containing neuropeptide Y, and (iii) neurons containing substance P. Each of these populations sends nerve fibres to the mucosa. Neuromedin U immunoreactivity is thus located in a variety of neurons serving different functions in the intestine and therefore probably does not have a single role in intestinal physiology.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Neurochemically similar myenteric and submucous neurons directly traced to the mucosa of the small intestine

Summary Antisera to neuropeptide Y (NPY) gave an intense immunohistochemical reaction of certain nerve cells in the myenteric and submucous plexuses of the guinea-pig small intestine. Each nerve cell had up to 20 branching, tapering processes that were less than 50 m long and a long process that could be followed for a considerable distance. This morphology corresponds to that of the type-III cells of Dogiel. The long process of each myenteric cell ran through the circular muscle to the submucosa, and in most cases the process could be traced to the mucosa. The submucous nerve cell bodies also had processes that extended to the mucosa. These cell bodies, in both plexuses, also stained with antisera raised against calcitonin generelated peptide (CGRP), cholecystokinin (CCK), choline acetyltransferase (ChAT) and somatostatin (SOM), but did not stain with antibodies against enkephalin, substance P or vasoactive intestinal peptide. Thus, it has been possible for the first time to trace the processes of chemically specified neurons through the layers of the intestinal wall and to show by a direct method that CGRP/CCK/ChAT/NPY/ SOM myenteric and submucous nerves cells provide terminals in the mucosa.     

Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine

A multidimensional chromatographic regimen has been used to isolate and purify a peptide showing immunoreactivity for neuromedin U from guinea pig small intestine. Microsequence Edman N-terminal analysis and C-terminal analysis by enzymatic digestion showed this peptide to be a nonapeptide with the following sequence: H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The C-terminal octapeptide of this sequence is the same as porcine NMU-8, and the C-terminal heptapeptide is identical to rat NMU(17-23).     

Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect

The occurrence and distribution of calcitonin gene-related peptide (CGRP) in the lower airways was studied by means of immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-like immunoreactivity (-LI) was observed in nerves from the epiglottis down to peripheral bronchi in rat, cat and guinea pig and also in human bronchi. Double staining revealed colocalization of CGRP-LI and substance P (SP)-LI in cell bodies of nodose and jugular ganglia as well as in axons and nerve terminals of the airways. Systemic capsaicin pretreatment induced a marked loss of the CGRP- and SP-immunoreactive (-IR) nerves in the lower airways. CGRP-IR was also present in epithelial endocrine cells and neuroepithelial bodies. The content of CGRP-LI as measured with RIA in guinea pig bronchi was significantly lower after capsaicin pretreatment. Analysis of human bronchial extracts revealed that CGRP-LI coeluted with synthetic human CGRP on HPLC. In the isolated perfused guinea pig lung capsaicin exposure caused overflow of CGRP-LI suggesting release from peripheral branches of sensory nerves. Both in vivo experiments in the guinea pig measuring insufflation pressure as well as in vitro studies on isolated guinea pig and human bronchi showed that whereas tachykinins contracted bronchial smooth muscle no contractile or relaxing effect was elicited by human or rat CGRP. However, CGRP caused relaxation of serotonin precontracted guinea pig and human pulmonary arteries. In conclusion, the presence and release of CGRP-LI from capsaicin sensitive nerves in the lower airways adds another possible mediator, in addition to tachykinins, of vascular reactions upon sensory nerve irritation.     

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.     

Occurrence of FMRF amide-like immunoreactive nerve fibers in guinea pig small intestine

We investigated the distribution of FMRF amide-like immunoreactivity in the small intestine of the guinea pig. Immunoreactive nerve fibers were found mainly in the myenteric and submucous plexuses and in the inner circular muscle layer. The labeled processes contained variable proportions of small clear vesicles 30-40 nm in diameter and large granular vesicles 80-120 nm in diameter. The large granular vesicles showed heavy immunoreactivity. The antisera against FMRF amide crossreact with peptides belonging to the pancreatic polypeptide family; it has therefore been suggested that the FMRF amide immunoreactivity demonstrated in the small intestine is caused by a peptide that is biosynthetically related to, but not necessarily a member of, the pancreatic polypeptide family.     

Isolation, structural characterization and pharmacological activity of dog neuromedin U

The neuromedin U-like immunoreactivity in an extract of dog small intestine was resolved by reversed-phase HPLC into two molecular forms. The primary structure of the larger form (NMU-25) was established as: Phe-Arg-Leu-Asp-Glu-Glu-Phe-Gln-Gly-Pro10-Ile-Ala-Ser-Gln-Val-Arg- Arg-Gln-Phe- Leu20-Phe-Arg-Pro-Arg-Asn-NH2. This sequence shows five substitutions relative to pig neuromedin U-25. The primary structure of the second peptide (NMU-8) was established as: pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The sequence contains the substitution pGlu for Tyr1 compared with pig neuromedin U-8. The potency of synthetic dog NMU-8 in contracting smooth muscle from the rat uterus (EC50 10 +/- 2 nM; mean +/- S.E., n = 6) was not significantly different from the corresponding potency of pig NMU-8 (EC50 16 +/- 5 nM) but the maximum response produced by the dog peptide was greater (58%; p less than 0.05) than that produced by pig NMU-8.     

Secretoneurin: A new peptide in the human enteric nervous system

Secretoneurin is a functional neuropeptide derived from secretogranin II (chromogranin C). This proprotein is processed to varying degrees in neuroendocrine tissues. In the present study we established by gel filtration high performance liquid chromatography that in human intestinal wall and mucosa an antiserum against secretoneurin detects as the major immunoreactive moiety the free peptide secretoneurin. In the mucosa some larger immunoreactive peptides were also present, however, a significant amount of the intact proprotein secretogranin II could not be detected. By immunohistochemistry we studied the distribution of secretoneurin within the gut. Antibodies to protein gene product 9.5 and chromogranin A were used to identify all neurons and endocrine cells, respectively, whilst those to the peptides substance P. CGRP and somatostatin were used for the further characterization of individual secretoneurin-positive structures. Secretoneurin immunoreactivity was found in nerve fibres in all layers of the gut wall. In both myenteric and submucous plexuses, nerve fibres and the majority of ganglion cells were secretoneurin-immunoreactive. In the mucosa, some secretoneurin-positive nerve processes ran parallel to the basal membrane of epithelial cells, occasionally invading the epithelial layer. Secretoneurin immunoreactivity was found in endocrine cells, mostly D cells, in the following regions in descending order of density: stomach/duodenum; rectum; colon; ileum. Thus, secretoneurin is a new major peptide within the human enteric neuroendocrine system. Its presence in abundant myenteric ganglion cells may imply a role in the modulation of gastrointestinal motility. The chemotactic properties of secretoneurin and its possible localization in sensory fibres suggest that this peptide may be involved in the genesis of intestinal inflammation.     

Localization of neurotensin-immunoreactive cells in the small intestine of man and various mammals.

Antibodies against synthetic bovine neurotensin were raised in rabbits and used to demonstrate neurotensin-immunreactive cells by immunohistochemical methods. In the jejunum and ileum of all species investigated (man, dog, monkey, cat, rabbit, sheep, rat, mouse, hamster, chinese hamster, gerbil, pig and guinea pig) cells were present in the mucosa, which reacted specifically with antineurotensin serum using the indirect immunofluorescence and peroxidase-antiperoxidase methods. In the monkey Tupaia the distribution of neurotensin-immunoreactive cells was examined by investigating serial sections through the entire gastro-entero-pancreatic (GEP) endocrine system, again showing most neurotensin-immunoreactive cells in the jejunum and ileum. The functional role of the presence of neurotensin immunoreactivity in the gut is discussed.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut

Summary The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut. An immunohistochemical study.

The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.