Substance P-like immunoreactive nerve fibers in the pars distalis of the anterior pituitary in the dog.

The pars distalis of the anterior pituitary is known to be regulated by hypothalamic hormones. Recently, we have discovered the presence of substance P-like immunoreactive nerve fibers in the pars distalis of the monkeys. Substance P-like immunoreactivity in the pars distalis of the dog was investigated in this study. A substantial amount of substance P-like immunoreactive nerve fibers with a large amount of varicosities were found. They were widely distributed in the gland, more abundant along its periphery. Most of them were closely related to the glandular tissue, some were located on vascular walls. Substance P-like immunoreactive nerve fibers were also found in the meningeal sheath of the anterior pituitary. They could be followed into the parenchyma of the gland.

Is the reduction of VIP the clue to the pathophysiology of Hirschsprung's disease?

The reduction of vasoactive intestinal peptide-(VIP) containing nerve fibres in the aganglionic segment in Hirschsprung's disease is thought to contribute to the sustained contraction of this intestinal segment. In order to study the significance of VIP in the pathogenesis of Hirschsprung's disease we used immunohistochemistry to evaluate the reduction of VIP-immunoreactive nerve fibres in aganglionic intestine compared to the ganglionic one. The VIP nerve fiber density was compared with the type of onset of disease (e.g. neonatal ileus or obstipation) and with the length of the aganglionic segment. No statistically significant correlation between these factors could be registered. This indicates a high complexity of the neuronal derangement in aganglionic intestine and that the degree of VIP deficiency alone does not correlate with the severity of the disease.

Altered inhibitory innervation of circular smooth muscle in Crohn's colitis. Association with decreased vasoactive intestinal polypeptide levels.

To determine whether decreased tissue vasoactive intestinal polypeptide levels might affect inhibitory neural input, fresh colonic specimens were obtained from patients with Crohn's colitis (n = 7) and normal subjects (n = 13). Immunoreactive vasoactive intestinal polypeptide levels were measured in the muscularis externa by radioimmunoassay and localized in tissue sections by immunostaining. Circular muscle strips were maintained in an organ bath; inhibitory junction potentials evoked by short- and long-duration field stimulation and resting membrane potentials were recorded using intracellular impalements. In Crohn's colitis, vasoactive intestinal polypeptide levels displayed a bimodal distribution in which 3 specimens had vasoactive intestinal polypeptide levels greater than or equal to 4 SE lower than the mean in normal specimens. In 3 specimens from Crohn's colitis with decreased vasoactive intestinal polypeptide levels, immunoreactive material was absent from the circular muscle layer and the myenteric plexus. Mean resting membrane potentials, mean amplitude of inhibitory junction potentials evoked by short-duration stimulation, and mean amplitude of initial inhibitory junction potentials evoked by long-duration stimulation were not different between the two groups. However, the mean amplitude of the 60th inhibitory junction potential during prolonged stimulation was decreased (p less than 0.01) in Crohn's colitis (6 mV) compared with normal specimens (11 mV). These results show that diminished neural input to circular muscle in Crohn's colitis was associated with decreased extractable vasoactive intestinal polypeptide levels and decreased staining of nerve fibers containing vasoactive intestinal polypeptide.

Terminations of LHRH-immunoreactive fibers in the subfornical organ of the opossum: an ultrastructural study.

Electron microscopic immunocytochemical approaches were used to analyze LHRH-containing elements in the subfornical organ of the opossum, a species in which this input to the subfornical organ is prominent. Not only were LHRH synaptic specializations easily demonstrated in the subfornical organ, forming axo-dendritic and axo-axonal contacts, but also LHRH-immunoreactive fibers contacted astrocytic end-feet on fenestrated capillaries and were found in the subependymal layer. LHRH-carrying elements in the subfornical organ may be important for relating reproductive functions to body fluid balance.

Acrolein depletes the neuropeptides CGRP and substance P in sensory nerves in rat respiratory tract.

The mammalian respiratory tract is densely innervated by autonomic and sensory nerves around airways and blood vessels. Subsets of these nerves contain a number of putative neurotransmitter peptides, such as substance P and calcitonin gene-related peptide (CGRP) in sensory nerves and vasoactive intestinal polypeptide (VIP), possibly serving autonomic functions. CGRP is also found in endocrine cells in rat airway epithelium. These peptides are all pharmacologically potent effectors of bronchial and vascular smooth muscle and bronchial secretion. Their functions in vivo are less well established. We have therefore examined the effects of inhaled acrolein, a sensory irritant, on three pulmonary neuropeptides: CGRP, substance P, and VIP. Groups of rats (n = 3 each) were exposed for 10 min to acrolein in air (Ct = 510, 1858, and 5693 mg.min/m3) or to air alone. Fifteen minutes later they were killed (pentabarbitone IP) and their respiratory tracts were dissected and fixed in 0.4% p-benzoquinone solution. Cryostat sections were stained by indirect immunofluorescence for a general nerve marker (PGP 9.5) and neuropeptides. The acrolein-treated animals had a dose-related decrease in tracheal substance P- and CGRP-immunoreactive nerve fibers compared with controls. No change was seen in total nerve fiber distribution and number (PGP 9.5) or VIP immunoreactivity, nor in CGRP-immunoreactive epithelial endocrine cells. It is concluded that the rat tracheal peptidergic nerves are a sensitive indicator of inhaled irritant substances. Their reduced immunoreactivity may be because of a release of sensory neuropeptides that could play a role in the physiological response to irritant or toxic compounds.

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).

Pregnancy reduces noradrenaline but not neuropeptide levels in the uterine artery of the guinea-pig.

Using histochemical, immunohistochemical and biochemical techniques, noradrenaline-, neuropeptide Y-, vasoactive intestinal polypeptide-, substance P- and calcitonin gene-related peptide-containing nerve fibres were studied in the uterine artery of virgin, progesterone-treated and pregnant guinea-pigs. Morphological changes following hormone treatment or in pregnancy were also evaluated in a quantitative study on semithin sections of the uterine artery. In late pregnancy, the number of noradrenaline-containing nerve fibres, which formed the densest plexus in virgin animals, was significantly decreased, a finding supported by a significant reduction in noradrenaline levels. This reduction was not mimicked by systemic progesterone treatment. In contrast, the innervation of the uterine artery by neuropeptide Y-containing nerve fibres was increased in pregnancy, while the other peptidergic nerves and peptide levels were unchanged after progesterone treatment and in pregnancy. These changes led to a predominance of innervation by neuropeptide Y- rather than noradrenaline-containing nerve fibres in late pregnancy. No morphological changes were detected following progesterone treatment, but pregnancy led to a marked increase in the cross-sectional area of the vessel accompanied by an increase in the thickness of the media.

Peptide-containing nerve fibers in the circumvallate papillae.

The occurrence and distribution of an array of neuropeptides and dopamine-beta-hydroxylase in the circumvallate papillae of monkey, pig, cow, ferret, cat, rat and mouse was studied by immunocytochemistry. The animals were chosen to represent species with different diets. Substance P/neurokinin A- and calcitonin gene-related peptide-containing fibers were numerous in the circumvallate papillae of all animals examined, with the highest frequency in monkey, pig, cow, rat and mouse; in ferret and cat moderate numbers were detected. Vasoactive intestinal peptide/peptide histidine isoleucine amide-containing fibers were numerous in the circumvallate papillae of pig, while they were moderate in number in monkey, ferret and mouse. Neuropeptide Y-containing fibers were few to moderate in number in the circumvallate papillae of all species. Galanin-containing fibers were numerous in the pig circumvallate papillae, while only a few fibers could be detected in monkey, cow, cat, rat and mouse. Somatostatin-containing fibers were seen only in the cat circumvallate papillae, gastrin-releasing peptide-containing fibers in the cow and cat, cholecystokinin/gastrin-containing fibers in the pig and cow. Dopamine-beta-hydroxylase-containing fibers were detected in all animals studied. They were few to moderate in number in the circumvallate papillae. There was no obvious link between the peptidergic innervation pattern and the food habits.

Vasoactive intestinal peptide-like immunoreactive nerve fibers in the pineal gland of the sheep.

Vasoactive intestinal peptide (VIP)-like immunoreactive nerve fibers were demonstrated by peroxidase antiperoxidase (PAP) immunohistochemistry to be distributed throughout the entire pineal gland of the sheep. VIP-containing fibers were observed along the blood vessels, penetrating into the gland from the pial capsule and also in the capsule itself. Some fibers left the perivascular position and entered the pineal parenchyma, where they were located among pinealocytes. This suggested that the VIPergic fibers might influence both pinealocytes and blood vessels of the gland. The location of VIP-containing fibers in the capsule of the pineal gland indicates that the fibers originate from perikarya located in a peripheral ganglion.

Three types of neurochemically defined autonomic fibres innervate the carotid baroreceptor and chemoreceptor regions in the guinea-pig.

The innervation of the carotid body, carotid sinus, and neighbouring arteries (common carotid artery; external carotid artery; occipital artery; ascending pharyngeal artery) was investigated in guinea-pigs by means of glyoxylic acid-induced catecholamine-fluorescence and immunohistochemistry using a variety of antisera against neuropeptides and tyrosine hydroxylase (TH). Fibres displaying catecholamine-fluorescence, TH- and neuropeptide Y-like immunoreactivity (NPY-LI) were less numerous in the carotid sinus than in all other arterial segments. Vasoactive intestinal polypeptide (VIP)-LI axons were almost lacking in the common carotid, external carotid and occipital arteries, consistently found in the carotid sinus, and more numerous in the ascending pharyngeal artery. Catecholaminergic, TH-, NPY- and VIP-LI fibres were observed deep in the media of the carotid sinus, where the baroreceptor terminals are located. In contrast, they did not enter the media in the adjacent arterial segments. All these fibres disappeared following excision of the superior cervical ganglion, but were unaffected by combined transection of the carotid sinus nerve and resection of the no-dose ganglion, suggesting a sympathetic origin. Double-staining immunofluorescence revealed at least three types of autonomic, presumably sympathetic fibres in the carotid sinus: 1) TH+/NPY+, 2) NPY+/VIP+, and 3) VIP+ fibres. This points to a non-noradrenergic efferent innervation of the carotid sinus in addition to the hitherto known noradrenergic sympathetic fibres. The three populations of autonomic fibres seen in the carotid sinus were also observed in the carotid body, but the paucity of NPY+/VIP+ double-labelled fibres raises doubt as to the functional significance of this particular fibre type in modulating arterial chemoreception. The multiplicity of neurochemically defined autonomic nerves to the carotid baro- and chemoreceptor regions probably reflects functionally separate pathways that are differently regulated and exert different effects.

A study of nerves containing peptides in the pulmonary vasculature of healthy infants and children and of those with pulmonary hypertension.

Nerves containing peptides that supply the human intrapulmonary vasculature were studied in 21 controls aged one month to 24 years and in 13 patients with pulmonary hypertension aged 11 days to eight years. An indirect immunofluorescence technique was used to study the distribution and relative density of nerve fibres containing the general neuronal marker, protein gene product 9.5; tyrosine hydroxylase; synaptophysin; neuropeptide tyrosine; vasoactive intestinal polypeptide; substance P, somatostatin; and calcitonin gene related peptide. At all ages in normal and hypertensive lungs neuropeptide tyrosine was the predominant neuropeptide associated with the pulmonary vascular nerves. In normal lungs the relative density of nerve fibres increased during childhood only in the arteries of the respiratory unit. Pulmonary hypertension was associated with the premature innervation of these arteries during the first year of life. Innervation of small, abnormally thick-walled pre-capillary vessels by predominantly vasoconstrictor nerves may help to explain the susceptibility of infants to pulmonary hypertensive crises.

Immunohistochemical localisation of a gastrin-releasing peptide-like material in area postrema, nucleus of the solitary tract and vagal motor nucleus in the brainstem of rat.

The presence of an endogenous gastrin-releasing peptide (GRP)-like peptide in the hindbrain of rat was demonstrated immunohistochemically using antisera directed against the N-terminus and C-terminus of GRP. N-terminal and C-terminal-like immunoreactive material were distributed throughout the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMV) and tractus solitarius (TS), as well as in areas postrema (AP) and substantia gelatinosa separating AP from NTS. Positive immunostaining was localised to a dense network of nerve fibres which project longitudinally along the neuraxis. Immunolabelled cell bodies were observed rostral to the obex, principally in the mediolateral subnucleus of NTS. These immunopositive neurones project their axons caudally and longitudinally towards the commissural subnucleus of the NTS. Immunolabelled cell bodies also were found in AP; they projected their axons caudally and ventrally towards NTS. Positive immunostaining was blocked by pre-adsorbing antisera with either GRP (1 nmol/ml) or bombesin (3 nmol/ml), but was unaffected by substance P (30 nmol/ml) and spared by capsaicin pretreatments which deplete sensory nerves of their peptide content. The results indicate that NTS neurons containing a GRP-like peptide connect the rostral and caudal regions of the dorsal vagal complex by way of longitudinal nerve tracts descending NTS and TS. Some neurons in AP also contain a GRP-like peptide and appear to connect with the dorsal vagal complex.

Immunohistochemical distribution and colocalization of regulatory peptides in the carotid body.

Current investigations on the immunohistochemical occurrence and co-occurrence of biogenic polypeptides in the mammalian carotid body were reviewed and extended by our own recent findings. The family of chromogranins and related peptides in glomus cells appears to have a widespread interspecies distribution, whereas other peptides investigated occur in a species-specific pattern. Immunoreactivity to antisera against opioids, which derive from the proenkephalin sequence, appears to be present in glomus cells of the rabbit, cat, dog, and a shrew. Conversely, glomus cells of pig and guinea pig predominantly are immunoreactive to cleavage products of prodynorphin, which co-occur in some cells with substance P and met-enkephalin-arg-phe, respectively. In the rat and Callithrix jacchus, opioid immunoreactivity is present in nerve fibres but not in glomus cells. Immunoreactivity to other peptides, such as neurotensin, cholecystokinin, neuropeptide Y, and galanin, is found only in one or two particular species. Neurotensin immunolabelling occurs in beagle dog glomus cells, which are known to lack substance P. Cholecystokinin immunoreactivity is present in glomus cells of dog and Callithrix, and co-exists with chromogranin A, neuropeptide Y, and substance P. Substance P appears to exist in both carotid body glomus cells and nerve fibres. Substance P immunoreactivity is present in glomus cells of all species investigated, except dog. Coexistence of substance P and calcitonin gene-related peptide (CGRP) is demonstrated in nerve fibres of the guinea pig carotid body, which originate in the petrosal and jugular ganglia. Other peptides visualized immunohistochemically in mammalian carotid body nerve fibres are vasoactive intestinal peptide and neuropeptide Y. The functional significance of the various peptides present in the carotid body is discussed.

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus).

Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities.(ABSTRACT TRUNCATED AT 250 WORDS)

[The role of immunoreactive nerves in the human bladder neck].

VIP-containing nerves have been considered as an inhibitory nerve of non-cholinergic, non-adrenergic action in the bladder neck. Using immunohistochemistry, VIP was stained in the bladder neck in 14 cases which involved 5 cases of bladder neck contracture in addition to 9 cases of benign prostatic hypertrophy (BPH). Consequently, the VIP immunoreactive nerve was not stained in the 4 cases of bladder neck contracture with a large quantity of residual urine which had the fibrosis of bladder neck, but in one case was stained without residual urine. In the BPH, 8 out of 9 cases were stained and they had small residual urine. It was fully stained in the cases without residual urine or with a small volume if they had it. The possible role of VIP in the bladder neck was supposed to be to maintain the bladder neck opening by the strong smooth muscle relaxation.

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

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.

Distribution of peptidergic terminals of enteric origin in the rat celiac ganglion.

We examined the distribution of enterofugal nerve terminals of bombesin-, cholecystokinin- and vasoactive intestinal polypeptide-like immunoreactivity in the rat celiac-superior mesenteric ganglion complex. The majority of these nerve terminals were concentrated in the mesenteric side of the ganglion. The present findings suggest that some functional specialization occurs in the celiac ganglion of the rat.

Topographic relations between tyrosine hydroxylase- and luteinizing hormone-releasing hormone-immunoreactive fibers in the median eminence of adult rats.

Employing electron microscopic double immunolabeling, we determined a close apposition of tyrosine hydroxylase (TH) and luteinizing hormone-releasing hormone (LHRH) nerve fibers in the rat median eminence (ME). These axo-axonic contacts occurred frequently in the internal and palisade zones, i.e. at the level of the fiber preterminals. In the superficial area of the ME, major TH fibers abutted on the basal lamina and some were projected into the pericapillary space of the portal vessels. Conversely, LHRH fibers were arrested by the endfeet of tanycytes in reaching the basal lamina.

Immunohistochemical characterization of the sheep suprachiasmatic nucleus.

Using 19 antisera raised against neuropeptides, amines or enzymes of amine biosynthesis, an immunohistochemical characterization of the sheep suprachiasmatic nucleus was performed. The most distinguishing characteristic of the sheep suprachiasmatic nucleus was the low density of serotonin- and neuropeptide Y-immunoreactive fibres; their concentration was similar to that in surrounding areas. This is different from observations in rodents but similar to those in primates. Moreover, the sheep suprachiasmatic nucleus is also characterized by a dense plexus of methionine-enkephalin-immunoreactive fibres. This has not been observed in other species. As in other species, such as rodents, the sheep suprachiasmatic nucleus contains numerous neurophysin-immunoreactive neurons and a few tyrosine hydroxylase-immunoreactive neurons. After colchicine pretreatment, many intensely stained vasoactive intestinal peptide-, vasopressin- and somatostatin-immunoreactive perikarya appeared, and more neurophysin-immunoreactive cell bodies were observed. Thus, although similarities exist among species, there are distinct differences in the neuro-chemical organization of the suprachiasmatic nucleus in the sheep and other species.

Distribution and localization of glial fibrillary acidic protein in colons affected by Hirschsprung's disease.

The distribution and localization of glial fibrillary acidic (GFA) protein were examined by means of immunohistochemistry in normoganglionic, oligoganglionic, and aganglionic segments of colons from 25 patients with Hirschsprung's disease, including four cases of long segment aganglionosis. In normoganglionic segments, GFA protein-positive glial cells were densely distributed within the myenteric plexus, but sparse in the submucous plexus. Aganglionic segments were completely devoid of glial cells with GFA protein immunoreactivity, coinciding with the lack of enteric ganglia. Instead, GFA protein was found specifically in association with the hypertrophic nerve fasciculi and their branches, which were mainly located in the intermuscular zone and submucosal connective tissue in the distal aganglionic segment of diseased bowels. However, two types of short and long segment aganglionosis differed in the distribution pattern of GFA protein; the extrinsic nerve fasciculi in short segment disease extended toward the normoganglionic segment, but in long segment disease they did not reach this area. A moderate number of GFA protein-positive fasciculi were observed within the circular muscle layer of proximal aganglionic and oligoganglionic parts in short segment aganglionosis, while no immunoreactive fasciculi were encountered within the circular muscle layer of the corresponding parts in long segment aganglionosis. Immunohistochemistry for GFA protein can be of excellent diagnostic value for the aganglionic colon with Hirschsprung's disease, since GFA protein immunohistochemistry discloses exclusively extrinsic, hypertrophic nerve fasciculi, characteristic of the bowel in cases of Hirschsprung's disease.