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.     

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.     

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide consisting of 38 amino acids (PACAP1–38) with a potent stimulatory action on adenylate-cyclase in rat pituitary. The presence of PACAP-like immunoreactivity in human brain was studied by radioimmunoassay. Co-localization of PACAP with arginine vasopressin and oxytocin was investigated by immunocytochemistry in the human hypothalamus. Immunoreactive PACAP was detected in all regions of human brain (cortex, thalamus, hypothalamus, pons and hemisphere of cerebellum) with the highest levels found in the hypothalamus (8.5±1.9 pmol/g wet weight, n=w, mean±S.E.M.). High performance liquid chromatography of the human hypothalamic ex approximately 50% of the immunoreactive PACAP was eluted in the position of PACAP1–38. Immunocytochemical studies showed the presence of PACAP immunoreactive neurons in the paraventricular and supraoptic nuclei of human hypothalamus. PACAP co-localized with arginine vasopressin in magnocellular cells of these nuclei. These findings suggest that PACAP1-38 plays important physiological roles in the human hypothalamus.     

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.     

In vivo effect of pituitary adenylate cyclase activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats.

In view of the recent demonstrations that pituitary adenylate cyclase activating polypeptide (PACAP) 38 stimulates the release of LH from superfused pituitary cells and that the hypothalamus and anterior pituitary have highly selective binding sites for the peptide, we have surveyed the effect of intraatrial injections of PACAP 38 and vasoactive intestinal peptide (VIP), which has 68% homology with PACAP 38, in intact adult male rats. Furthermore the effect of intracerebroventricular (icv) injection of PACAP 38 was investigated. Intraatrial (10, 30, 100 micrograms) and icv (8, 32 micrograms) administration of PACAP 38 stimulated LH release significantly (P less than 0.01) in a dose-related fashion. Icv injection at a dose of 0.8 microgram was ineffective. The time course pattern of LH release by intraatrial injection and that by icv injection was similar, but the LH levels increased by intraatrial injection were much higher than that by icv injection. Intraatrial administration of VIP had almost no effect on LH release. These findings suggested that PACAP 38 stimulates LH release in vivo.     

PACAP—A Multifacetted Neuropeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated from ovine hypothalamus based on its ability to stimulate cAMP production in pituitary cell cultures. The peptide exists in two forms, both of which are derived from the same precursor. PACAP38 and the C‐terminal truncated PACAP27 can interact with three subtypes of receptors activating adenylate cyclase and/or phospholipase C. Since its discovery, numerous studies have provided evidence that PACAP is a pleiotropic substance having a broad spectrum of biological functions; the peptide can act as a hormone, neurohormone, autocrine/paracrine substance, neurotransmitter, neuromodulator, neurotrophic factor, and immunomodulator. Two examples of the functional role of PACAP on the biological timing system are presented: 1) the transient expression of PACAP during the periovulatory period in ovarian cells, in which PACAP functions as an autocrine/paracrine inducer of progesterone secretion and subsequent luteinization; and 2) the role of PACAP as a neurotransmitter in the retinohypothalamic tract mediating photic regulation of the brain's biological clock.     

PACAP--a multifacetted neuropeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated from ovine hypothalamus based on its ability to stimulate cAMP production in pituitary cell cultures. The peptide exists in two forms, both of which are derived from the same precursor. PACAP38 and the C-terminal truncated PACAP27 can interact with three subtypes of receptors activating adenylate cyclase and/or phospholipase C. Since its discovery, numerous studies have provided evidence that PACAP is a pleiotropic substance having a broad spectrum of biological functions; the peptide can act as a hormone, neurohormone, autocrine/paracrine substance, neurotransmitter, neuromodulator, neurotrophic factor, and immunomodulator. Two examples of the functional role of PACAP on the biological timing system are presented: 1) the transient expression of PACAP during the periovulatory period in ovarian cells, in which PACAP functions as an autocrine/paracrine inducer of progesterone secretion and subsequent luteinization; and 2) the role of PACAP as a neurotransmitter in the retinohypothalamic tract mediating photic regulation of the brain's biological clock.     

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.     

PACAP—A Multifacetted Neuropeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated from ovine hypothalamus based on its ability to stimulate cAMP production in pituitary cell cultures. The peptide exists in two forms, both of which are derived from the same precursor. PACAP38 and the C-terminal truncated PACAP27 can interact with three subtypes of receptors activating adenylate cyclase and/or phospholipase C. Since its discovery, numerous studies have provided evidence that PACAP is a pleiotropic substance having a broad spectrum of biological functions; the peptide can act as a hormone, neurohormone, autocrine/paracrine substance, neurotransmitter, neuromodulator, neurotrophic factor, and immunomodulator. Two examples of the functional role of PACAP on the biological timing system are presented: 1) the transient expression of PACAP during the periovulatory period in ovarian cells, in which PACAP functions as an autocrine/paracrine inducer of progesterone secretion and subsequent luteinization; and 2) the role of PACAP as a neurotransmitter in the retinohypothalamic tract mediating photic regulation of the brain's biological clock.     

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.     

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 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.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the expression of PACAP in cultured tilapia astrocytes

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone-releasing hormone (GHRH)-PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP(38) and tGHRH and a short form (166 bp) encoding only tPACAP(38). The short form was found to be more abundant in astrocytes. Addition of ovine PACAP(38) (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP(38) at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC(1)-R) mRNA in the astrocytes was not responsive to PACAP(38) treatment. The tPACAP(38) expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP(38) expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.     

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.     

Chemical characterization of vasoactive intestinal polypeptide-like immunoreactivity in ovine hypothalamus and intestine.

Two forms of pituitary adenylate cyclase activating polypeptides with 38 (PACAP38) and 27 residues (PACAP27) respectively were recently isolated from ovine hypothalamic tissues. The N-terminal 28 amino acids sequence of PACAP was found to have 68% homology with porcine vasoactive intestinal peptide (VIP). In order to determine whether the primary structure of VIP of ovine hypothalamus is identical with porcine VIP or similar to PACAP, VIP immunoreactivity as determined by radioimmunoassay for porcine VIP was isolated in a pure form from ovine hypothalamic extracts. VIP was also isolated from ovine intestine. Amino acid analysis as well as amino acid sequence analysis showed that ovine hypothalamic and intestinal VIP were identical to porcine VIP, but different from PACAP.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.     

Neuronal interaction between melanin-concentrating hormone- and alpha-melanocyte-stimulating hormone-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.