VIP and PHM and their role in nonadrenergic inhibitory responses in isolated human airways

There is increasing evidence in many species that vasoactive intestinal peptide (VIP) may be a neurotransmitter in nonadrenergic inhibitory nerves. We have studied the effect of electrical field stimulation (EFS), exogenous VIP, and isoproterenol (Iso) on human airways in vitro. We have also studied a related peptide, peptide histidine methionine (PHM), which coexists with VIP in human airway nerves, and in separate experiments studied fragments of the VIP amino acid sequence (VIP1-10 and VIP16-28) for agonist and antagonist activity. Human airways were obtained at thoracotomy and studied in an organ bath. In bronchi EFS gave an inhibitory response that was unaltered by 10(-6) M propranolol but was blocked by tetrodotoxin, whereas in bronchioles there was little or no nonadrenergic inhibitory response. VIP, PHM, and Iso all caused dose-dependent relaxation of bronchi, VIP and PHM being approximately 50-fold more potent than Iso. VIP, but not Iso, mimicked the time course of nonadrenergic inhibitory nerve stimulation. In contrast bronchioles relaxed to Iso but not to VIP or PHM. Neither propranolol nor indomethacin altered the relaxant effects of VIP or PHM, suggesting a direct effect of these peptides on airway smooth muscle. Neither of the VIP fragments showed either agonist or antagonist activity. We conclude that VIP and PHM are more potent bronchodilators of human bronchi than Iso and that the association between the relaxant effects of these peptides and nonadrenergic inhibitory responses suggests that they may be possible neurotransmitters of nonadrenergic inhibitory nerves in human airways.     

Evidence for common precursors but differential processing of VIP and PHM in VIP-producing tumors

To elucidate the biosynthesis of vasoactive intestinal polypeptide (VIP) and investigate the suggestion that the prepro-VIP contains another peptide designated PHM (the peptide with N-terminal histidine and C-terminal methionine amide) in its sequence, the concentration and molecular forms of immunoreactive VIP and PHM in 14 human VIP producing tumors (VIP-omas) were determined. Elevated quantities of both peptides were found in all tumor extracts but the concentration of PHM did not correlate with that of VIP and the ratio VIP/PHM varied from 0.5 to 8.5. Gel chromatography showed that in addition to peaks corresponding to VIP and PHM, two larger molecular forms with Kd values of 0.31 and 0.36 which displayed both VIP and PHM immunoreactivity were present. While the proportions between the various PHM molecular forms varied considerably, the relative contribution of the VIP immunoreactive peaks was rather constant from tumor to tumor. The molecular pattern was unaffected by protein denaturing with guanidine hydrochloride and cleavage of sulfide bonds with dithiothreitol. The findings indicate that VIP and PHM are co-produced in VIP-omas probably from common larger molecular forms and that differences in the post-translational processing between tissues exist.     

Characterization and Regional Distribution of Peptides Derived from the Vasoactive Intestinal Peptide Precursor in the Normal Human Brain

To study the biosynthetic processing of the precursor for vasoactive intestinal peptide (prepro-VIP) in the human brain, we have developed antisera against the five functional domains of the precursor molecule: prepro-VIP 22-79, peptide histidine methionine (PHM), prepro-VIP 111-122, VIP, and prepro-VIP 156-170. The antisera were used in radioimmunoassays in combination with HPLC to identify and quantify the peptides in regions of the human brain. All five peptides were expressed, but mainly in nonequimolar ratios. In only three regions were the same amounts of VIP and PHM found; in the remaining areas the concentration of PHM was two-thirds that of VIP. The concentrations of prepro-VIP 22-79, prepro-VIP 111-122, and prepro-VIP 156-170 were considerably lower than the corresponding VIP concentrations, and the relative concentration of prepro-VIP 111-122 differed between cortical and subcortical areas. A small proportion of the VIP precursor followed a pathway in which the dibasic conversion site after PHM is not cleaved, as evidenced by the presence of a C-terminally extended form of PHM. Finally, it was found that the C-terminal lysine residue of prepro-VIP is not removed during processing. The findings indicate that differences in the posttranslational processing of prepro-VIP exist in subpopulations of neurons in the human brain.     

Cosecretion of peptide histidine methionine (PHM) and vasoactive intestinal peptide (VIP) in patients with VIP-producing tumors

Regional specific antibodies and chromatography were used to analyze the concentration and molecular forms of vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in plasma from 39 patients with VIP-producing tumors. Plasma VIP concentrations ranged from 29 to 2550 pmol/l and the corresponding PHM immunoreactive values measured with C-terminally directed antibody were 42 to 2100 pmol/l which correlated closely with the VIP concentrations. N-terminal PHM concentrations were significantly higher than the C-terminal values ranging from 92 to 5850 pmol/l and correlated poorly with the corresponding VIP concentrations. Infusion experiments with PHM disclosed that the higher levels of N-terminal immunoreactivity could not be explained by slower metabolic clearance or by degradation to smaller N-terminal immunoreactive forms. N-terminally directed PHM antibody revealed, in addition to intact PHM, a larger immunoreactive form in patient plasma which constituted the major proportion of the total immunoreactivity. In conclusion, VIP and PHM are cosecreted from VIPomas and measurement of PHM, especially N-terminal immunoreactivity, may be useful in this condition.     

Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland

The effects of the neuropeptides VIP, PHM and substance P (SP) on vascular smooth muscle tone, K+ secretion from exocrine elements and tissue content of cyclic AMP (cAMP) in the human submandibular gland were studied in vitro. All three peptides caused relaxation of noradrenaline contracted human submandibular arteries at nM concentrations. SP was slightly more active than VIP and PHM which had a similar potency as vasodilators. Only carbachol but not VIP, PHM or SP stimulated K+ secretion from exocrine elements of the human submandibular gland. Principally similar in vitro effects on K+ secretion were obtained on the cat submandibular gland, but in the rat not only carbachol but also SP stimulated K+ secretion. VIP and PHM increased cAMP production of exocrine elements in the human submandibular gland in nM concentrations. VIP was about 5-fold more potent than PHM with regards to cAMP production. In conclusion, VIP, PHM and SP relaxed human submandibular arteries in vitro. Both VIP and PHM stimulated cAMP production in glandular tissue but none of the three peptides induced K+ secretion from human submandibular gland tissue. This suggests that, in contrast to the situation in the rat, SP does not cause watery salivation in man, while VIP and PHM may modulate protein e.g. amylase content of the saliva.     

Peptide histidine methionine (PHM) increases ileostomy output

The human vasoactive intestinal peptide (VIP) gene also encodes peptides histidine methionine (PHM) which has substantial sequence homology with VIP. Both are present in nerve fibers in the human ileum and circulate in greatly increased concentrations in patients with the watery diarrhoea syndrome. We have infused PHM (23 pmol/kg/min) into 5 patients with ileostomies to determine the effect of PHM on human ileal output. Plasma PHM levels rose from 22 +/- 6 to 6013 +/- 874 pM (mean +/- S.E.M.) during PHM infusions and ileal output rose from 16 +/- 3 to 177 +/- 27 g/30 min (P less than 0.0001). PHM infusions also produced a significant fall in the percentage of solid material and a rise in the concentration of chloride in the ileal effluent. Mean plasma PHM concentrations during PHM infusions were equal to the highest levels seen in patients with the watery diarrhoea syndrome, so PHM may contribute to diarrhoea in this condition. Neuronal PHM may exert physiological control over ileal transport of water and electrolytes.     

PACAP, a VIP-like peptide, in neurons of the esophagus.

The lower esophagus of guinea-pig, cat, sheep and man was analyzed for pituitary adenylate cyclase activating peptide (PACAP), a novel vasoactive intestinal peptide (VIP)-like peptide, using immunocytochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were numerous in the longitudinal and circular muscle layers of sheep and man, moderate in numbers in cat, while being few in the esophagus of guinea-pig. A few PACAP-immunoreactive nerve cell bodies and numerous nerve fibers were seen in the myenteric ganglia of the esophagus of cat, sheep and man. In the lower esophagus of cat, sheep and man all PACAP-containing nerve cell bodies and nerve fibers stored VIP. The results of radioimmunoassay of PACAP in extracts of specimens from man were in good agreement with the immunocytochemical findings. High performance liquid chromatography revealed one major peak of PACAP-like immunoreactivity in extracts of human esophagus. We suggest that neuronal PACAP may serve to modulate motor activity and secretion in the lower esophageal sphincter region.     

Neuropeptides in the female genital tract: effect on vascular and non-vascular smooth muscle

The presence of vasoactive intestinal polypeptide (VIP), substance P (SP), somatostatin, enkephalin, and avian pancreatic polypeptide (APP) in nerves in the female genital tract raises the question of their physiological significance as neurotransmitter substances. We have examined the effect of these peptides on non-vascular uterine smooth muscle in vivo as well as in vitro, and the effect on blood flow in the genital tract of rabbit and cat. SP caused a dose-dependent increase in mechanical and myoelectrical activity, an action which could be antagonized by VIP. Substance P, leu-enkephalin and VIP induced a concentration related increase in blood flow of the uterus, where VIP seems to be the most potent vasodilator. Neither the effects on vascular nor on non-vascular smooth muscle were inhibited by adrenergic nor cholinergic blocking agents. APP was able to inhibit the VIP-induced vasodilation in rabbits. These findings suggest that several peptides are involved in the local nervous control of both uterine contractions and haemodynamic events.     

Non-amidated forms of VIP (glycine-extended VIP and VIP-free acid) have full bioactivity on smooth muscle

The aim of the present study was to elucidate the importance of the C-terminal amide group for the biological activity of vasoactive intestinal peptide (VIP). Two synthetic peptides lacking the amide group: VIP having a carboxyl group at the C-terminus and the intermediate biosynthetic precursor, glycine-extended VIP were compared with VIP itself regarding the ability to inhibit spontaneous activity in smooth muscle strips from rat stomach and human Fallopian tube. Both the glycine-extended VIP and VIP having a carboxyl group at the C-terminus caused a significant and dose-dependent inhibition of smooth muscle activity and displayed dose-response curves similar to VIP. The potencies of the VIP variants did not differ significantly from that of VIP. Thus, alpha-carboxyamidation of VIP is not a prerequisite for biological activity.     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

Levels of neural vasoactive intestinal polypeptide in rat uterus are markedly changed in association with pregnancy as shown by immunocytochemistry and radioimmunoassay

Immunocytochemical studies have shown that the rat uterus is well innervated by nerve fibers containing vasoactive intestinal polypeptide (VIP). The fibers were associated with both vascular and nonvascular smooth muscle cells, and they were somewhat more numerous in the cervix compared to the uterine horns. This was confirmed in radioimmunologic determinations. Pregnancy induced a marked, almost 50% reduction in the total content of VIP in the uterine horns, which was associated with an almost complete disappearance of immunocytochemically visible nerve fibers in this part of the uterus. The innervation normalized within 25 days following delivery. Less marked changes occurred in the VIP innervation of the cervical region, where the concentration of the peptide was reduced mainly as a result of the increased tissue weight during pregnancy.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide and secretin from the ovine small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucinamide (PHI) and secretin were separated and purified to homogeneity from ovine small intestine, using radioimmunoassay and radioreceptor assay for detection. An efficient and rapid purification sequence included acid extraction, concentration on a bulk C18 cartridge, filtration on a Fractogel column, ion-exchange chromatography on Mono-S and a maximum of three successive reverse-phase HPLC steps. The amounts of peptides obtained from 450 g wet weight tissue were 20 micrograms VIP, 15 micrograms PHI and 5 micrograms secretin. The as yet unknown amino acid sequences of the three peptides were found to be identical to those of the corresponding bovine peptides.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

The use of region-specific antibodies in the characterization and localization of vasoactive intestinal polypeptide-like substances in the rat gastrointestinal tract

Antisera specific for different regions of porcine VIP have been used in radioimmunoassay and immunohistochemical studies of immunoreactive VIP in rat small and large intestine. Cation exchange chromatography of intestinal extracts separated two major and one minor peak of immunoreactivity. One major peak eluted in a similar position to natural porcine VIP and was read equally by NH₂-terminal-specific, and mid- and COOH-terminal-specific antisera. A second major peak, and the minor peak, eluted earlier than porcine VIP, and were read significantly less well with mid- and COOH-terminal antisera compared with NH₂-terminal-specific antisera. All forms of VIP occurred mainly in extracts of muscle layers of the gut, and no antiserum revealed more than trace amounts of immunoreactivity in mucosal extracts. In immunohistochemical studies all antisera demonstrated fluorescent nerve fibres in the enteric plexuses, circular smooth muscle and lamina propria; some antisera demonstrated nerve cell bodies predominantly in the submucous plexus. NH₂-terminal-specific antisera also demonstrated a sparse population of mucosal endocrine-like cells in the ileum and colon that were not seen with other antisera. It is concluded that VIPergic neurons of the rat gut contain a peptide closely resembling porcine VIP and at least two less basic variants with similar NH₂-terminal antigenic determinants. VIP-like peptides may also occur in endocrine cells, but since these peptides appearto fact that the majority of neuronal VIP in rat gut exists in a form that is both chromatographically and immunochemically distinct from porcine VIP, and may well possess different biological properties.     

Expression of preproVIP-derived peptides in the human gastrointestinal tract: a biochemical and immunocytochemical study

In order to study biosynthetic processing of the precursor for vasoactive intestinal peptide (preproVIP) in the human gut we have developed antisera against the five functional domains of the precursor molecule: preproVIP 22-79, peptide histidine methionine (PHM), preproVIP 111-122, VIP and preproVIP 156-170. The antisera were used to quantify and characterize VIP-precursor peptides by radioimmunoassay (RIA) together with high-pressure liquid Uchromatography (HPLC) and to examine their cellular localization and colocalization by immunocytochemistry. All five peptides were expressed but not in equimolar amounts as expected from the amino acid sequence of the precursor. However, the ratios between them were fairly constant throughout the gastrointestinal tract. The only exceptions were the lower concentrations of PHM and preproVIP 111-122 in the gastric antrum which could be explained by the presence of PHV (the C-terminally extended form of PHM which includes preproVIP 111-122) in large concentrations in this region. It was also found that the C-terminal lysine residue of preproVIP is not removed during processing. Immunocytochemically all preproVIP-derived peptides were shown in neuronal elements. They had a similar distribution throughout the gut suggesting coexistence, a finding which was supported by doublestaining. The findings indicate that differences in the posttranslational processing of preproVIP exist in subpopulations of neurons in the human gut.     

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

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.     

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.     

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.