Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a similar structure to that of vasoactive intestinal peptide (VIP) and both the polypeptides belong to the same molecular group, the secretin-glucagon superfamily. PACAP and VIP have possible potency as hypothalamic factors mediating the release of pituitary hormones in the fish pituitary. However, the roles of PACAP and VIP in the central nervous systems of fish have not yet been made clear. Recently, it was reported that PACAP and/or VIP are involved in the feeding behavior of the mouse and chick. Therefore, we investigated the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic PACAP and VIP on food intake in the goldfish, Carassius auratus. Cumulative food intake was significantly decreased by ICV injection of PACAP (11 or 22 pmol/g body weight) or VIP (11 or 22 pmol/g) during a 60-min observation period after treatment. IP administration of PACAP (44 or 88 pmol/g) or VIP (22 or 44 pmol/g) induced a significant decrease in food intake during a 60-min observation period after treatment. These results suggest that PACAP and VIP may be involved as feeding regulators in goldfish.     

Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity

Pituitary adenylate cyclase-activating polypeptide (PACAP) interacts with three types of PACAP/VIP-receptors. The PAC1-receptor accepts PACAP as a high affinity ligand but not vasoactive intestinal peptide (VIP) similarly binding to VPAC1- and VPAC2-receptors. To identify those amino acids not present in VIP defining PAC1-receptor selectivity of PACAP, radio receptor binding assays on AR4-2J cells were performed. It could be shown that PACAP(1-27) exhibited a distinct and much higher susceptibility to VIP-amino acid substitutions, compared to PACAP(1-38). Positions 4 and 5 seem to be most important for receptor binding of PACAP(1-27), whereas position 13 was identified to be crucial for maximal affinity of PACAP(1-38). PACAP(29-38) extension analogues of VIP revealed a stabilizing effect of the C-terminus of PACAP(1-38) on the optimal peptide conformation. The substitution analogues were also checked for their capacity to stimulate IP3 and cAMP formation in AR4-2J cells. Compared to PACAP(1-27) and PACAP(1-38), most analogues revealed potencies reduced congruously to their lower binding affinities. However, one of the analogues, PACAP(1-27) substituted in position 5, may represent a weak antagonist since this peptide was less potent in inducing second messengers than in label displacement. Our findings indicate that PACAP(1-27) and PACAP(1-38) differ in terms of their requirement of the amino acids in positions 4, 5, 9, 11 and 13 for maximal interaction with the PAC1-receptor.     

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.     

Relationship between anorexigenic action of pituitary adenylate cyclase-activating polypeptide (PACAP) and that of corticotropin-releasing hormone (CRH) in the goldfish, Carassius auratus

Our recent research has indicated that intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake and locomotor activity in the goldfish. However, the anorexigenic mechanism of PACAP has not yet been clarified. The aim of this study was to investigate the relationship between the anorexigenic action of PACAP and that of corticotropin-releasing hormone (CRH), which is implicated in the regulation of energy homeostasis as a powerful anorexigenic peptide in the goldfish brain. We first examined feeding-induced changes in the expression of CRH mRNA, and the effect of ICV administration of PACAP on the expression of CRH mRNA in the goldfish brain. Semiquantitative analysis revealed that the expression of CRH mRNA was significantly increased by excessive feeding for 7 days. ICV administration of PACAP at a dose sufficient to suppress food intake induced a significant increase in the expression of CRH mRNA. We also examined the effect of alpha-helical CRH(9-41), a CRH antagonist, on the anorexigenic action of PACAP in the goldfish. The inhibitory effect of PACAP was completely suppressed by treatment with alpha-helical CRH(9-41). We finally investigated the effect of ICV-administered CRH on locomotor activity in the goldfish. CRH at a dose sufficient to suppress food intake induced a significant increase in locomotor activity, unlike ICV-injected PACAP. These results suggest that, in the goldfish, the anorexigenic action of PACAP is related to the CRH neuronal pathway, but that the modulation of locomotor activity by PACAP is independent of modulation by CRH.     

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 (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.     

Effect of pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) on tissue oxygen content--treatment in central nervous system of mice

It has been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in preventing neuronal cell death and is also a potent vasodilator. Cerebral hypotension and hypoperfusion during cerebral ischemia and neurodegenerative diseases are well known as some of the negative factors which aggravate neuronal cell death. Nevertheless, the effect of PACAP on the cerebral circulation was not understood well. Therefore, in the present study, we determined the mean arterial blood pressure (MBP), regional cerebral blood flow (rCBF) and cerebral oxygen content (pO2) in mice, and estimated the therapeutically useful doses of PACAP. Under barbiturate anesthesia, polyethylene tubes were inserted into mice to monitor MBP and to administer PACAP (5 x 10(-13)-5 x 10(-8) mol/kg) or vasoactive intestinal peptide (VIP; 5 x 10(-12) and 5 x 10(-9) mol/kg). Then, MBP, rCBF and cerebral pO2 were simultaneously measured in the mice. PACAP (5 x 10(-10)-5 x 10(-9) mol/kg) injections transiently decreased MBP, and cerebral pO2. PACAP (5 x 10(-8) mol/kg) injections produced a long-lasting potent decline of MBP, rCBF and cerebral pO2. Therefore, PACAP should be applied at low doses which do not influence the MBP and cerebral circulation to determine the therapeutically useful doses of PACAP for neuroprotection.     

Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is produced by hypothalamic neurons which terminate within the median eminence suggesting that it may be a hypophysiotropic hormone. However, little endocrine activity has been ascribed to the peptide. Therefore we studied the effects of PACAP on prolactin (Prl) release from dispersed cultivated rat pituitary cells in vitro using conventional cultures as well as the reverse hemolytic plaque assay (RHPA). Furthermore the effects of the peptide on in vitro GH release were assessed. In addition, the activity of the peptide on in vivo release of Prl and GH was studied in hypothalamus-lesioned animals. PACAP dose dependently inhibited Prl release form dispersed pituitary cells in both, monolayer cell cultures and the RHPA, whereas GH secretion was not affected. In hypothalamus-lesioned rats which have high Prl levels due to the absence of hypothalamic dopamine, PACAP further stimulated Prl release. Serum GH increased more than 20 fold in response to the intravenous PACAP infusion. Thus in vitro (inhibition of Prl release, no effect on GH release) and in vivo (stimulation of both hormones) experiments yielded contradicting effects of PACAP on pituitary hormone release. We suggest that PACAP may stimulate the release of a paracrine, yet unknown factor which in the intact pituitary overrides the direct inhibitory action of PACAP on the lactotropes. The same or another paracrine factor may also enhance in vivo GH release. In cell culture the paracrine factor is diluted by the medium. Therefore the peptide never reaches effective concentrations which are present within the intact pituitary tissue.     

Immunohistochemical observation of pituitary adenylate cyclase-activating polypeptide (PACAP) and adenohypophysial hormones in the pituitary of a teleost, Uranoscopus japonicus

A neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) has possible potency as a hypothalamic factor mediating the release of pituitary hormones, especially growth hormone (GH), in the fish pituitary. We used double-immunostaining to examine the relationship between PACAP nerve fibers and adenohypophysial hormone-producing cells in the pituitary of a teleost, the stargazer Uranoscopus japonicus, and enzyme immunoassay to determine the quantity of PACAP in the stargazer brain, in conjunction with the body mass and gonad somatic index (GSI) of fish. In adult stargazer, PACAP-like immunoreactive (PACAP-LI) nerve fibers and endings were identified in both the neurohypophysis and adenohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as prolactin, somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. PACAP-LI nerve fibers were also identified close to immunoreactive GH cells in the pituitary of young fish. The concentration of immunoreactive PACAP in whole brain ranged from 100 to 800 pmol/g wet weight, in fish with weighing 70-480 g. A negative correlation was found between the concentration of immunoreactive PACAP in the whole brain and body weight, but there was no relation between the former and GSI. These results suggest that PACAP may act as a hypophysiotropic factor in the stargazer pituitary.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象, 垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造砀ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细     

Discovery and SAR of hydrazide antagonists of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor type 1 (PAC1-R)

Potent small molecule antagonists for the PAC(1)-R have been discovered. Previously known antagonists for the PAC(1)-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.     

Receptor-independent cellular uptake of pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP), a hypophysiotropic neurohormone, participates in the regulation of pleiotropic functions. The recent discovery of intracellular PACAP receptors in the brain and the testis as well as the physico-chemical characteristics of PACAP, i.e. extended α-helix containing basic residues, prompted us to evaluate the propensity of PACAP to cross the plasma membrane in a receptor-independent manner. Using confocal microscopy and flow cytometry, we demonstrated the ability of FITC-conjugated PACAP to efficiently penetrate into the internal cell compartment by direct translocation and endocytosis through clathrin-coated pits and macropinocytosis. Our study also revealed that, once inside the cells, PACAP38 is not entirely degraded by intracellular enzymes and that a significant amount of intact PACAP38 is also able to exit cells. Moreover, using binding assay on rat nuclear fractions from various tissues, PACAP nuclear receptors were identified. We also found that PACAP stimulates calcium release in rat testis nuclei. Interestingly, PACAP27 and PACAP38 but not VIP were able to upregulate de novo DNA synthesis in testis nuclei and that this effect was abolished by PACAP(6-38). These results support the presence of PAC1 receptors at the nuclear membrane and raise questions about their role in the biological activity of the peptide. These findings contribute to the characterization of PACAP as an intracrine factor and suggest that these intracellular PAC1 binding sites, probably associated with specific biological activities, should be taken into account during the development of PACAP-based drugs.     

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

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

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