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.     

Immunocytochemical analysis of calcitonin gene-related peptide and vasoactive intestinal polypeptide in Merkel cells and cutaneous free nerve endings of cats

Summary Calcitonin gene-related peptide (CGRP)-and vasoactive intestinal polypeptide (VIP)-immunoreactivity were observed to coexist in Merkel cells of cats. No differences in peptide content were found between Merkel cells located in epithelia of the hard palate, in hairy and glabrous skin of the upper lip, and in vibrissae follicles. CGRP-and VIP-immunoreactive nerve fibres were also found near CGRP/VIP-immunoreactive Merkel cells. In the vibrissae follicles some CGRP-and VIP-immunoreactive nerve terminals end abutting on the glassy membrane. Other CGRP immunoreactive nerve fibres penetrate the epithelium of the skin and end within it. Electron microscopy of vibrissae follicles revealed that Merkel cell neuntes are not immunostained and that immunostained nerve fibres form unmyelinated bundles before ending freely. Thus, CGRP-and VIP immunoreactive nerve fibres in cat skin do not end as Merkel cell neuntes but as different kinds of free nerve endings.     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

Cellular localization and ontogeny of immunoreactive Vasoactive intestinal polypeptide (VIP) in the chicken gut

Summary Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves were abundant along the entire digestive tract of the chicken. In the proventriculus, gizzard and small intestine VIP nerves were numerous around glands and less numerous in the smooth muscle. Submucosal blood vessels were often encircled by VIP nerves. VIP nerves were also seen in the submucosal and myenteric plexus. In the large intestines the VIP innervation of the smooth muscle was more predominant, while there was a rather sparse supply of VIP nerves around the base of the crypts. This innervation pattern was a consistent finding with four different VIP antisera. VIP-immunoreactive cells, however, were demonstrated with only three of the antisera. They were found scattered in the epithelium of the proventriculus and small and large intestines. The failure of one of the antisera to demonstrate endocrine cells suggests that the VIP-immunoreactive material in these cells differs from that in nerves. Conceivably, the material present in nerves represents VIP, while that in endocrine cells represents cross-reacting peptides or other molecular forms of VIP.VIP nerves appeared comparatively early in embryonic development. They appeared in the upper part of the digestive tract at 13 days of incubation and in the colon a few days before hatching; at this stage, only smooth muscle received VIP nerves. The adult pattern of innervation was established about two to four weeks after hatching. VIP-immunoreactive endocrine cells appeared in the intestines a few days before hatching. The adult frequency of occurrence was established about one week after hatching.     

Vasoactive intestinal polypeptide-like immunoreactivity in nerves of the pancreatic islet of the teleost fish,Gillichthys mirabilis

Summary In the teleost fish, Gillichthys mirabilis, vasoactive intestinal polypeptide (VIP)-like immunoreactivity is present in some nerve fibres of the principal pancreatic islet and surrounding tissues, the vagus and splanchnic nerves, the coeliac ganglion and the wall of the intestine. The nerves of the pancreatic islet that contain VIP-like immunoreactivity probably correspond to one of the two types of non-cholinergic, non-adrenergic (peptidergic) nerve previously described in this fish. Similarities in the localisation of hormonal peptides in fish and mammals suggest that the regulation of gastroenteropancreatic physiology in fish may resemble that of mammals.This work was supported by grants from the Medical Research Council of Great Britain and the National Institutes of Health, USA (Grant No. AM 17161)     

Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man

Summary The occurrence and origin of substance P (SP)-immunoreactive (IR) nerves in the lower respiratory tract was studied by means of immunohistochemistry in the guinea-pig, rat, cat and man. In addition, biopsies from human material were also analysed by radioimmunoassay. SP-IR nerves were seen in four principal locations: 1) under or within the lining epithelium, 2) around blood vessels, 3) within the bronchial smooth muscle layer, and 4) around local tracheobronchial ganglion cells. Ligation experiments combined with capsaicin pretreatments indicated that all SP-IR nerves in the respiratory tract are sensory. The trachea seems to be mainly supplied by the vagal nerves, while intrapulmonary bronchi and blood vessels receive SP-IR nerves of both vagal and non-vagal (spinal) origin. SP-IR nerves were also found in the human bronchi with principally similar location as in the guinea-pig. The levels of SP-IR in the trachea and peripheral bronchi of man were about 3–4 pmol/g, which is in the same range as the content of corresponding tissues from the guinea-pig.In conclusion, the present experimental findings of SP-IR nerves in the lower respiratory tract in both experimental animals and man support the functional evidence for the importance of SP in the vagal and non-vagal (spinal) control of bronchial smooth muscle tone and vascular permeability.     

Comparative distribution of neuropeptide tyrosine-,vasoactive intestinal polypeptide-, substance P-immunoreactive,acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat

Summary Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive (cholinergic) and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.     

The origin of VIP-containing nerves in the urinary bladder of rat

The innervation of the urinary bladder is known to include a considerable number of nerves containing vasoactive intestinal polypeptide (VIP). The origin of such nerves in the bladder of rat was investigated in this study using the methods of immunocytochemistry and radioimmunoassay combined with surgical sectioning of the hypogastric and/or pelvic nerves to the bladder. Eight days after pelvic nerve sectioning proximal to the main pelvic ganglion, VIP-immunoreactive nerves and VIP content were markedly increased from the level in the sham-operated rat bladder. Sectioning of hypogastric or both nerve pathways led to a less significant increase. It was therefore postulated that the majority of VIP-immunoreactive nerves originate from ganglia located either close to the bladder or within the bladder wall. It is interesting that in these experiments the VIP content of the bladder nerves is inversely related to the changes in motility that would be expected to result from the nerve sections.     

Immunohistochemical localization of vasoactive intestinal polypeptide(VIP)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Summary The localization of vasoactive intestinal polypeptide (VIP) in the hypothalamus of the quail has been studied by means of light- and electron-microscopic immunohistochemistry. Numerous VIP-immunoreactive perikarya are distributed in the caudal portion of the nucleus infundibularis (n. tuberis) and nucleus mamillaris lateralis, and sparse in the preoptic area, nucleus supraopticus and nucleus paraventricularis. Dense localization of immunoreactive-VIP fibers is observed in the external layer of the median eminence, in close contact with the primary portal capillaries. The main origins of these fiber terminals are VIP-immunoreactive perikarya of the nucleus infundibularis. These neurons are spindle or bipolar and extend one process to the ventricular surface and another to the external layer of median eminence. They are CSF-contacting neurons and apparently constitute the tubero-hypophysial tract that links the third ventricle and the hypophysial portal circulation. VIP-reactive neurons in the nucleus mamillaris lateralis also project axons to the external layer of the median eminence, constituting the posterior bundle of the tuberohypophysial tract. Numerous VIP-immunoreactive perikarya occur also in the nucleus accumbens/pars posterior close to the lateral ventricle. They are also CSF-contacting neurons extending a process to the lateral ventricle. There are moderate distributions of VIP-reactive fibers in the area ventralis and in the area septalis.Ultrastructurally, the immunoreactive products against VIP are found in the elementary granules, 75–115 nm in diameter, within the nerve fibers in the median eminence.This investigation was supported by Scientific Research Grants No. 00556196, No. 56360027 and No. 56760183 from the Ministry of Education of Japan to Professor Mikami and Mr. Yamada     

The distribution and cellular origin of vasoactive intestinal polypeptide in the avian gastrointestinal tract and pancreas

Summary The distribution and origins of vasoactive intestinal peptide (VIP) in the gut and pancreas of the turkey were studied by radioimmunoassay of tissue extracts and by immunocytochemistry. Several antisera were used that vary in their specificity for different regions of porcine or chicken VIP. Radioimmunoassays using NH₂-terminal specific antisera that react almost equally with porcine and chicken VIP's revealed significant amounts of immunoreactive VIP in extracts of pancreas, brain and all regions of the gastrointestinal tract from crop to colon. Highest concentrations (300pmol/g) were found in the colon muscle, and concentrations were generally low (< 20 pmol/g) in the mucosal layers of the small intestine. After ion exchange chromatography of extracts on CM-Sephadex three immunoreactive forms of VIP were separated corresponding to the three molecular forms previously found in mammalian gut extracts. In immunocytochemical studies nerve fibres were found throughout the gut, and in the pancreas. Immunoreactive nerve cell bodies were also identified in the submucous plexus throughout the gut, but were particularly prominent in the oesophagus and pancreas. It has previously been shown that VIP is a strong stimulant of the flow of pancreatic juice in birds whereas the structurally related hormone secretin, which is known to control the flow of pancreatic juice in mammals, is a weak stimulant. It is proposed that in birds VIP might regulate the pancreas, and other aspects of gut function, as a neurotransmitter or neurohormone.     

Immunohistochemical localization and vascular effects of vasoactive intestinal polypeptide in skeletal muscle of the cat

Summary Scattered vasoactive intestinal polypeptide (VIP) — immunoreactive nerves were found in the striated muscle of the hind limb of the cat, where they usually were associated with small blood vessels. VIP-immunoreactive nerves were also demonstrated in the sciatic nerve; after nerve ligation an abundance of intensely immunoreactive VIP fibres were seen proximal to the ligation. Intraarterial infusion of VIP into the isolated hind limb of the cat had dramatic effects on different sections of the vascular bed. Thus, VIP dilated the resistance vessels leading to a marked increment in muscle blood flow. VIP also relaxed the capacitance vessels causing regional pooling of blood; it increased the capillary surface area available for fluid exchange. Infusions of VIP at a dose of 8 g/min significantly inhibited the vasoconstriction induced by electrical stimulation of the regional sympathetic nerves. It is suggested that local nervous release of VIP may act as a modulator of vascular tone in skeletal muscle.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Neuropeptides in the gut: quantification and characterization of cholecystokinin octapeptide-, bombesin- and vasoactive intestinal polypeptide-like immunoreactivities in the myenteric plexus of the guinea-pig small institute

The localization of CCK8-, bombesin- and VIP-like immunoreactivities in the myenteric plexus of the guinea pig small intestine has been studied by radioimmunoassay of extracts of longitudinal muscle strips obtained with and without adherent myenteric plexus; concentrations were compared with those in other regions of the gut. In innervated strips of longitudinal muscle of ileum there was approximately 14 pmol/g CCK8-, 32 pmol/g bombesin- and 135 pmol/g VIP-like immunoreactivity; concentrations were reduced by over 70% in denervated strips. Gel filtration and ion exchange chromatography indicated that over 80% of CCK immunoreactivity was due to CCK8; no evidence was found of significant amounts of smaller COOH-terminal fragments. Bombesin immunoreactivity occurred in two forms, the major one resembling the amphibian tetradecapeptide in its elution from gel filtration columns. Immunoreactive VIP differed markedly from porcine VIP in immunochemical and chromatographic properties; the data suggest that guinea pig VIP is less basic than porcine VIP and that the two peptides differ in structure in their NH2-terminal regions. Some functional implications of these findings are discussed.     

Vasoactive intestinal polypeptide (VIP) immunoreactivity of endocrine-like cells in the feline pyloric mucosa

Summary Immunoreactivity to VIP by endocrine-like cells in the feline pyloric mucosa was examined by using three kinds of region-specific anti-porcine VIP sera. VIP-immunoreactive endocrine-like cells were detected clearly with all of the VIP antisera used. They were located mainly around the neck of the pyloric glands. Some of these endocrine-like cells showed dilution-dependent immunoreactivity against VIP antisera. The immunostaining intensity of VIP-immunoreactive endocrine-like cells showing dilution-independence could not be distinguished from those of nerve elements. The present results suggest that the immunoreactivity with properties very similar to those of authentic VIP may be present in the endocrine-like cells of the feline pyloric glands.     

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.     

Spontaneous neurite outgrowth and vasoactive intestinal peptide-Iike immunoreactivity of cultures of human paraganglioma cells from the glomus jugulare

Summary The chief cells of paraganglionic tissues have morphological and functional similarities to adrenal chromaffin cells, and both cell types are derived from the neural crest. In the present investigation cells from two glomus jugulare paragangliomas were studied in culture. Approximately 50% of the cells from one tumor, and 7% from the other spontaneously formed neurite-like processes. Numerous granular and agranular synaptic-like vesicles also appeared in the process-forming cells. In contrast to findings with normal and neoplastic adrenal chromaffin cells, addition of nerve growth factor (NGF) to the culture medium had no major effects on proportion of cells with processes. Dexamethasone caused only a small decrease in process length. Culturing of the tumors also appeared to promote production of material with VIP-like immunoreactivity. It is concluded that the phenotype of paraganglioma as well as pheochromocytoma cells may be altered in vitro. Responsiveness to specific factors such as NGF or steroids, however, may vary for related tumor cell types in different anatomic locations.     

On the origin and distribution of vasoactive intestinal polypeptide-, peptide HI-, and cholecystokinin-like-immunoreactive nerve fibers in the rat iris

Summary The presence and distribution of nerve fibers expressing immunoreactivity to the neuropeptides vasoactive intestinal polypeptide, peptide HI and cholecystokinin was examined in stretch-prepared rat iris whole mounts. By use of antiserum to vasoactive intestinal polypeptide an irregular, relatively sparse network of varicose, intensely fluorescent fibers was observed innervating both the dilator plate and the sphincter area. Positive fibers were present also in the ciliary body and the choroid membrane. Surprisingly, a large variation in the amount of vasoactive intestinal polypeptide-positive nerves was seen among irides. Furthermore, an uneven distribution of fluorescent nerve fibers was observed within individual irides. Thus, some areas had a relatively dense innervation, whereas others were devoid of immunoreactive nerve fibers. A similar fiber system was detected using antiserum to peptide HI. In all probability, vasoactive intestinal polypeptide and peptide HI coexist within the same nerve population. A denser and more regular network of cholecystokinin-positive fibers was found in normal rat irides. Such fibers were also present in the sphincter area and in high density in the choroid membrane. Neither extirpation of the superior cervical nor the ciliary ganglion caused any detectable decrease in amount of either vasoactive intestinal polypeptide/peptide HI- or cholecystokinin-positive fibers. However, capsaicin, which in the iris causes permanent disappearance of substance-P fibers, had a similar effect on cholecystokinin-positive fibers, whereas no effect was noted on the vasoactive intestinal polypeptide/peptide HI fiber network. It is concluded that the rat iris contains a network of vasoactive intestinal polypeptide/peptide HI-positive nerves that does not originate in either the superior cervical or the ciliary ganglion, and most probably also not in the trigeminal ganglion, and a cholecystokinin-positive network that probably originates in the trigeminal ganglion.     

Localization of VIP-immunoreactive nerves in airways and pulmonary vessels of dogs,cats, and human subjects

Summary VIP-containing neurons were localized in lungs from dogs, cats, and human subjects by means of the indirect immunofluorescence technique. Nerve fibers and terminals were observed in the smooth muscle layer and glands of airways, and within the walls of pulmonary and bronchial vessels, especially at the medial-adventitial junction. VIP-positive nerve cell bodies were identified in ganglia located in the walls of bronchi. These findings provide an anatomic basis for the possible modulation of airway and pulmonary vascular function by this neuropeptide.Supported by National Research Service Award HL 5914 and by Lung Center Award HL 14187 from NHLBI, USPHS     

Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.