Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development

Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.     

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.     

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.     

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.     

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.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

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.     

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.     

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.     

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.     

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.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.     

High-level expression, purification, and characterization of the recombinant grass carp pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP(38)) is a potent secretagog for growth hormone and gonadotropin in fish species. To obtain recombinant grass carp PACAP(38), its open reading frame was subcloned in pET32a(+) vector to express thioredoxin (Trx)-PACAP fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the thioredoxin-PACAP reached 36% of the total proteins, and more than 85% of fusion protein existed as soluble form. Using Ni(2+)-chelating affinity chromatography, 102 mg of Trx-PACAP(38) with a purity of 97% was obtained from 342 mg of crude proteins from a 1-liter culture of Escherichia coli. The purified Trx-PACAP specifically inhibited T98G human glioblastoma cell proliferation, but the fusion partner had no effect in this regard. Moreover, this inhibition was totally abolished by PACAP-specific antibody.     

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     

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.