Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

Summary Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the gastrointestinal tract of the rat: distribution and effects of capsaicin or denervation

The expression of pituitary adenylate cyclase activating polypeptide (PACAP) was studied in the gastrointestinal tract (GI-tract) of normal rats using radioimmunoassay, chromatography, immunocytochemistry, and in situ hybridization. PACAP-38, PACAP-27, and PACAP-related peptide were demonstrated in all parts of the GI-tract, PACAP-38 being the predominant form confirmed by chromatography. PACAP-immunoreactive nerve fibers and nerve cell bodies were found in the myenteric ganglia throughout the GI-tract. PACAP-containing nerve cell bodies were also demonstrated in the submucous ganglia of the small and large intestine. The synthesis of PACAP in intrinsic neurons was confirmed by in situ hybridization. Double immunostaining showed that PACAP is present in calcitonin gene-related peptide-containing sensory nerve fibers as well as in vasoactive intestinal polypeptide (VIP)- or VIP/gastrin-releasing peptide (GRP)-containing (intramural) nerve fibers in the upper GI-tract and in anally projecting, intrinsic VIP-and VIP/nitric oxide syntase-containing nerve cell bodies and nerve fibers in the small and large intestine. Neonatal treatment with capsaicin significantly reduced the concentration of PACAP-38 in the esophagus, stomach, and colon. Extrinsic denervation decreased the PACAP-38 concentration in the stomach, while no change was observed in the small intestine. These results indicate that PACAP- immunoreactive nerve fibers in the GI-tract originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources suggesting that PACAP may have diverse gastrointestinal functions.     

A novel neuropeptide,pituitary adenylate cyclase-activating polypeptide (PACAP), in human intestine: evidence for reduced content in Hirschsprung's disease

Summary A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exhibits sequence homology with vasoactive intestinal polypeptide (VIP) and occurs in the mammalian brain, lung and gut. The distribution of PACAP in ganglionic and aganglionic portions of the large intestine of patients with Hirschsprung's disease was examined by immunohistochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were distributed in all layers of the ganglionic and aganglionic segments of the intestine, although they were less numerous in the latter, and PACAP-immunoreactive nerve cell bodies were seen in the ganglionic portion of the intestine. The concentration of immunoreactive PACAP was lower in the aganglionic than in the ganglionic segment of the intestinal wall. PACAP and VIP were found to coexist in both ganglionic and aganglionic segments of the intestine. Apparently, PACAP participates in the regulation of gut motility. The scarcer PACAP innervation of the aganglionic segment may contribute to the defect in intestinal relaxation seen in patients with Hirschsprung's disease.     

Helospectin-like immunoreactivity in the esophagus

Helospectin I and II are two non-amidated, VIP-like peptides, isolated from the salivary gland venom of the lizard Heloderma horridum. The lower esophagus of cat, sheep and man was analyzed for helospectin-like immunoreactivity.

Immunocytochemistry revealed helospectin-immunoreactive nerve fibers in the muscle layers, submucosa and mucosa of all species studied. In myenteric ganglia helospectin-immunoreactive nerve fibers and nerve cell bodies could be seen. Double immunostaining for helospectin and vasoactive intestinal peptide (VIP) revealed their coexistence in nerve fibers and cell bodies throughout the lower esophagus of all species tested. Double immunostaining for helospectin and neuropeptide Y revealed their coexistence in nerve fibres surrounding vascular and non-vascular smooth muscle. In the cat and sheep (but not in man) a subpopulation of the helospectin/VIP-containing fibers stored, in addition, substance P.

The helospectin-immunoreactive material in the esophagus probably constitutes a novel neuropeptide. The distribution of the VIP/helospectin-immunoreactive neurons and fibers indicates their possible involvement in the regulation of motor and secretory activities.     

PACAP (pituitary adenylate cyclase-activating peptide)-like immunoreactivity in the gill arch of the goldfish, Carassius auratus: distribution and comparison with VIP

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalamus. It is present in neuronal elements of a number of peripheral organs. We have examined whether PACAP occurs in the gill arch of Carassius auratus L. in which our recent studies have shown the presence of VIP-like peptide. Immunohistochemistry has revealed PACAP-like immunoreactivity in the anterior branches of the post-trematic glossopharyngeal and vagus nerves. PACAP-immunoreactive nerve cell bodies and fibers are present in connective tissue on the oral side of the gill arch. Colocalization studies carried out by the application of double immunofluorescence show that a PACAP-like peptide coexists with VIP in the same nerve cell bodies and fibers. The localization pattern of PACAP in the gill arch of goldfish suggests its possible involvement in the regulation of secretory activities.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the rat mammary gland

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP) family of peptides, is present in the brain and in neuronal elements of a number of peripheral organs. Since no information on PACAP in the mammary gland exists, we have investigated, by radioimmunoassay and immunohistochemistry, the occurrence and distribution of PACAP immunoreactivity in the mammary gland of lactating and non-lactating rats. A specific monoclonal mouse anti-PACAP antibody'has been used to show that the peptide is located in nerve fibres associated with bundles of circular and longitudinal smooth muscle surrounding the lactiferous duct of the nipple. PACAP-immunoreactive nerve fibres and nerve bundles are present in the subepidermal connective tissue of the nipple and in the mammary parenchyma, some of the fibres being in close contact with blood vessels. Occasionally, a few delicate varicose fibres are associated with secretory alveoli and lactiferous ducts. The majority of PACAP-positive nerve fibres are, however, located in the glabrous skin of the nipple and the hairy skin adjacent to the nipple forming a subepithelial plexus from which delicate varicose nerve fibres enter the overlying epithelium. Double immunostaining for PACAP and a marker for sensory neurons, calcitonin gene-related peptide, has disclosed that the two peptides are almost completely co-localized. A minor population of the PACAP-immunoreactive nerve fibres shows co-existence with VIP. Although no obvious changes at the immunohistochemical level could be observed during pregnancy or lactation, elevated concentrations of immunoreactive PACAP-38 in mammary extracts have been found during lactation. Our data suggest that PACAP is involved in the nervous control of mammary gland function, probably in the transmission of suckling stimuli.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.     

Pituitary adenylate cyclase-activating polypeptide in intrinsic and extrinsic nerves of the rat pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.     

Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

Pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres were demonstrated in the rat pineal gland. These fibres entered the pineal gland through the conarian nerve at the distal tip of the gland. A high density of the fibres was observed in the capsule of the gland, from where the immunoreactive elements penetrated into the pineal perivascular spaces and parenchyma. The majority of PACAP-immunoreactive nerve fibres also contained calcitonin gene-related peptide (CGRP). Some PACAP-immunoreactive nerve fibres contained neuropeptide Y (NPY), but only occasionally was PACAP colocalized with vasoactive intestinal peptide (VIP). After removal of both superior cervical ganglia, a high number of PACAP-containing nerve fibres were still present in the gland. In the nervous system PACAP is present in two isoforms, PACAP-38 and PACAP-27. The concentration of PACAP-38 in the superficial pineal gland was determined by radioimmunoassay to be 20.4 pmol/g tissue at midday and 18.9 pmol/g tissue at midnight. The concentration of PACAP-27 was only about 3% of the concentration of PACAP-38. In summary, this study is the first demonstration of a PACAP-containing innervation of the rat pineal gland. The PACAP concentration in the pineal gland does not exhibit a day-night difference. The colocalization of PACAP with calcitonin gene-related peptide in the pincalopetal nerve fibres indicates that the majority of PACAP-immunoreactive nerve fibres might originate from the trigeminal ganglion.     

Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus

A 38 residue neuropeptide was recently isolated from ovine hypothalamus in our laboratory, and named pituitary adenylate cyclase activating polypeptide (PACAP38) based on its biological activity. Rabbit antisera against synthetic PACAP27 were characterized by ELISA for immunohistochemical use. PACAP-immunoreactive neuronal elements having similar distributions were demonstrated in both human and spider monkey hypothalami. Many PACAP-immunoreactive cell bodies were present in the supraoptic and paraventricular nuclei. Immunopositive nerve fiber networks were stained throughout the hypothalamus, including in both external and internal zones of the tuber cinereum, close to the transition of the pituitary stalk (median eminence). These results suggest that PACAP plays multifunctional roles as a hypophysiotropic hormone, neurohypophysial hormone, neurotransmitter or neuromodulator in higher vertebrate species including man.     

Distribution of peptidergic nerves in the choroid plexus, focusing on coexistence of neuropeptide Y, vasoactive intestinal polypeptide and peptide histidine isoleucine

Choroid plexus from rat, guinea-pig, rabbit and pig was investigated by light-microscopic immunohistochemistry and by radioimmunoassay for the presence of neuropeptides. A moderately dense supply of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), respectively, was found around blood vessels and in close relation to the secretory epithelium in both pig and rabbit, while lower densities of nerve fibers were found in rat and guinea-pig. Peptide concentrations ranged from 10-40 pmolequivalents/g (pmoleqv/g) for NPY and 0.5-6 pmoleqv/g for VIP in all four species. Peptide histidine isoleucine (PHI) immunoreactive nerve fibers were present in pig choroid plexus at a lower density than NPY and VIP but with a similar distribution. Low concentrations of substance P (0.3-3 pmoleqv/g) and calcitonin gene-related peptide (0.1-3 pmoleqv/g) were found to a varying degree in choroid plexus tissue from the different species, while immunohistochemical investigation was unable to detect any immunoreactive nerve fibers. NPY was often found to coexist with VIP and PHI in pig choroid plexus, while a lesser amount of nerve fibers showed coexistence of NPY and the noradrenaline synthetizing enzyme, dopamine-beta-hydroxylase. Surgical sympathetic denervation by excision of the superior cervical ganglion in the rabbit abolished NPY-containing nerve fibers, as revealed by immunohistochemistry, but only decreased NPY levels by one third, which may be due to different identity of the peptide being detected by the two techniques. It is concluded that NPY-containing nerve fibers have a dual origin in the choroid plexus and coexist with either noradrenaline or VIP/PHI.     

Pituitary adenylate cyclase-activating polypeptide: localization and differential influence on isolated hearts from rats and guinea pigs

This study was done to determine if pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive nerve fibers occur in cardiac muscle as well as intracardiac ganglia of rats and guinea pigs and to clarify the chronotropic actions of PACAP27 in the same species using isolated heart preparations. PACAP nerve fibers were not detected in atrial or ventricular muscle of rat or guinea pig but a few stained nerve fibers occurred in the atrioventricular bundle of the guinea pig. Stained nerve fibers were prominent in intracardiac ganglia of both species. PACAP27 caused a dose-dependent tachycardia in isolated rat hearts (+39 +/- 3 beats/min with 1 nmol, n = 6). Positive and/or negative chronotropic responses were evoked by PACAP27 in guinea pig heart, depending on dose and prior exposure to the peptide. PACAP27 also caused arrhythmias in several guinea pig hearts. Treatment with atropine eliminated or prevented PACAP-evoked bradycardia and arrhythmias, implicating cholinergic neurons in these responses. Positive chronotropic responses to PACAP were unaffected by beta-adrenergic receptor blockade in either species, suggesting that tachycardia resulted from a direct action on the heart. These observations support the conclusion that endogenous PACAP could have a role in regulating parasympathetic input to the heart but through different mechanisms in rats versus guinea pigs. A direct positive chronotropic influence of endogenous PACAP is unlikely since atrial muscle lacks PACAP-immunoreactive nerve fibers.     

Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas.

The distribution of calcitonin gene-related peptide (CGRP), substance P/tachykinin (SP/TK), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and gastrin-releasing peptide (GRP) immunreactivities (IR) in the rat pancreas was investigated using radioimmunoassay and immunohistochemistry. CGRP, NPY and VIP tissue contents are much higher than GRP and SP/TK concentrations. Peptide-containing nerves are distributed to both the exocrine and endocrine pancreas. However, differences exist in terms of density and targets of innervation for each peptidergic system. In the acini and through the stroma, fibers IR for CGRP, NPY and VIP are greater than GRP- and SP/TK-containing processes. The vasculature is supplied by a prominent NPY, CGRP and, to a lesser extent, SP/TK innervation. VIP-IR is found occasionally, and GRP-IR is never detected, in fibers associated with blood vessels. Around ducts, CGRP- and NPY-positive neurites are greater than SP/TK- greater than or equal to VIP-IR fibers, whereas GRP-containing nerves are not visualized. In the islets, the density of peptidergic nerves is: VIP-, GRP- greater than or equal to CGRP-IR greater than NPY or SP/TK. In intrapancreatic ganglia. VIP- and, to a lesser extent, NPY-IRs are found in numerous neuronal cell bodies and in nerve fibers; GRP-IR is present in numerous nerve processes and in few cell bodies; CGRP- and SP/TK-IRs are detected only in fibers wrapping around unlabeled ganglion cells. The majority of CGRP-IR fibers contain SP/TK-IR. The existence of differential patterns of peptidergic nerves suggests that peptides exert their effects on pancreatic functions via different pathways.     

The distribution and origin of VIP in the spinal cord of six mammalian species

The distribution of VIP-immunoreactivity was studied in the spinal cord and dorsal root ganglia of 6 mammalian species. Immunoreactive fibres and cell bodies were most apparent in the dorsal horn, dorsolateral funiculus, intermediolateral cell columns and the area around the central canal. The distribution of VIP immunoreactivity was similar in all species studied, mouse, rat, guinea pig, cat, horse and the marmoset monkey. There were fewer VIP fibres in the dorsal horn of cervical and thoracic segments than in lumbosacral segments. Using radioimmunoassay this gradient increase was quantitatively most marked in the sacral spinal cord of the cat. In dorsal root ganglia few nerve cell bodies but numerous fibres were present. A dual origin for VIP in the spinal cord is suggested: (A) Extrinsic, from dorsal root afferent fibres since immunoreactivity was decreased in dorsally rhizotomized animals (cats and rats) and in capsaicin pretreated rats (microinjection of dorsal root ganglia). (B) From local cell bodies intrinsic to the spinal cord which became visible after colchicine pretreatment of rats.     

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.     

Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity in the ureter of the duck

The presence, distribution and colocalisation of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity have been studied in the duck ureter by using Western blot analysis, radioimmunoassays (RIA) and immunohistochemistry. The presence of both PACAP-38 and PACAP-27 was demonstrated, PACAP-38 being the predominant form. PACAP-immunoreactive fibres and neurons were found in all the ureteral layers. Double immunostaining showed that PACAP was almost completely colocalised with vasoactive intestinal peptide (VIP). Moreover, PACAP was found in substance P (SP)-containing ureteral nerve fibres and in SP-containing dorsal root ganglion neurons. RIA performed on denervated ureters demonstrated that almost half of the ureteral PACAP was extrinsic in origin. These findings suggest that, in birds, PACAP has a role in diverse nerve-mediated ureteral functions.     

Innervation of the rat pineal gland by PACAP-immunoreactive nerve fibers originating in the trigeminal ganglion: a degeneration study

In order to establish that the pineal gland is innervated by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibers originating in the trigeminal ganglion, ophthalmic and maxillary nerves were transected by using a subtemporal fossa approach. The number of PACAP-immunoreactive nerve fibers in the pineal gland of rats with a total transection of the nerve was compared with that of rats without surgery. In the operated rat, PACAP-immunoreactive nerve fibers in the superficial pineal decreased remarkably, indicating that the trigeminal ganglion was the origin of these nerve fibers. This research provides evidence supporting the hypothesis that PACAP-immunoreactive nerves regulate the synthesis and/or secretion of melatonin in the pineal gland.     

Nitric oxide synthase-containing, peptide-containing, and acetylcholinesterase-positive nerves in the cat lower oesophagus

Summary The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE-positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility.     

Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man

By immunohistochemistry it was found that PHI- and VIP-like immunoreactivity (-IR) occurred in the same autonomic neurons in the upper respiratory tract, tongue and salivary glands with associated ganglia in rat, guinea-pig, cat, pig and man. VIP- and PHI-like immunoreactivity was also found in similar locations in the human heart. The N-terminally directed, but not the C-terminally directed, PHI antiserum or the VIP antiserum stained endocrine cells in the pig duodenum. This suggests the existence of an additional PHI-like peptide. Ligation of nerves acutely caused marked overlapping axonal accumulations of PHI- and VIP-IR central to the lesion. Two weeks after transection of the nerves, both types of immunoreactivities were still observed in accumulations both in the axons as well as in the corresponding cell bodies. The levels of PHI- and VIP-IR in normal tissues from the cat were around 10-50 pmol/g with a molar ratio of about 1 to 2. Systemic administrations of PHI and VIP induced hypotension, probably due to peripheral vasodilation in both guinea-pig and cat. Furthermore, both PHI and VIP caused an inhibition of the vagally induced increase in respiratory insufflation pressure in guinea-pig. PHI and VIP relaxed the guinea-pig trachea in vitro, suggesting a direct action on tracheobronchial smooth muscle. VIP was about 5-10 times more potent than PHI with regard to hypotensive effects and 2-3-fold, considering respiratory smooth muscle-relaxant effects in the guinea-pig. PHI was about 50-fold less potent to induce hypotension in the cat than in the guinea-pig. Although species differences seem to exist as regards biological potency, PHI should also be considered when examining the role of VIP as an autonomic neurotransmitter.