Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.     

Regulation of gene expression in PC12 cells via an activator of dual second messengers: pituitary adenylate cyclase activating polypeptide.

In this study we demonstrate that the activator protein-1 (AP-1) DNA motif, initially considered to be unresponsive to cyclic AMP (cAMP), does function as a cAMP-response element in PC12 cells. A luciferase reporter gene driven by the collagenase promoter that contains the AP-1 motif is responsive to cAMP as well as phorbol esters when transfected in PC12 cells. We have recently shown that pituitary adenylate cyclase activating peptide (PACAP) has neurotrophic properties and activates both adenylylcyclase and the inositol lipid cascade in PC12 cells. Consistent with these actions, we demonstrate that PACAP is an effective activator of luciferase reporter genes whose promoters bear the AP-1 motif, as well as the related DNA element that binds the protein CREB. Both the cAMP and inositol lipid pathways appear to play a role in the activation of these motifs by PACAP. Mutation of the AP-1 motif and its juxtaposition to a heterologous promoter proves that the AP-1 motif is a locus for response to cAMP and PACAP. The luciferase reporter genes bearing the AP-1 motif are not cAMP responsive in HeLa tk- cells, indicating that the mode of second-messenger responsiveness is cell-type specific.     

Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide with 38 (PACAP38) or 27 (PACAP27) amino acid residues, structurally related to vasoactive intestinal peptide (VIP). Bovine brain membrane has a PACAP specific receptor interacting with both PACAP27 and PACAP38. Affinity-labeling of the receptor with [125I]PACAP27 identified a dominant band of Mr = 60 k. The labeling density of the 60 k band decreased in the presence of unlabeled PACAP27 or PACAP38, whereas the 60 k band remained in the presence of unlabeled VIP. Binding of [125I]PACAP27 to the membrane decreased in the presence of GTP and the labeling density of the 60 k band decreased concomitantly. The results indicate that bovine brain has a specific PACAP receptor, whose apparent molecular weight is 57 k (substracting the molecular weight of [125I]PACAP27 from 60 k).     

Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide

In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as     

Anorectic and neurochemical effects of pituitary adenylate cyclase activating polypeptide in rats

Pretreatment of rats with intrahypothalamic injections of pituitary adenylate cyclase activating peptide (PACAP) 10 min prior to the injection of neuropeptide Y (NPY) significantly reduced food and water intake during the 4-h measurement period. Intrahypothalamic injection of PACAP in schedula-fed rats also reduced food and water intake for 2 h. A smaller 1-h reduction of water intake was observed in water-deprived rats, suggesting that the anticonsummatory effects of PACAP were primarily against food intake. PACAP treatment did not alter hypothalamic concentration of NPY, nor were neurotransmitters, precursors, or metabolites altered substantially in corpus striatum or nucleus accumbens regions. These results demonstrate primary anorectic effects of intrahypothalamic injection of PACAP. The demonstration of these anorectic effects may suggest a role of cyclic AMP activation and inhibition in the control of satiety and hunger.     

Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion.

A novel neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), which has been isolated from ovine hypothalami, shows 68% homology with vasoactive intestinal peptide (VIP). Since VIP stimulates amylase secretion from the pancreas, we investigated the effect of PACAP and VIP on rat pancreatic exocrine secretion after intravenous injections of PACAP-27, PACAP-38, or VIP at doses of 2.5, 5 or 10 nmol/kg. Results showed: 1) Bolus injection of PACAP stimulated pancreatic amylase and protein secretions in a dose-dependent manner; and 2) Stimulation of amylase secretion with 10 nmol/kg of PACAP-27 was greater than that induced with the same dose of VIP or PACAP-38 (p less than 0.05).     

Copper(II) complexation by pituitary adenylate cyclase activating polypeptide fragments.

Results are reported from potentiometric and spectroscopic (UV-Vis, CD, and ESR) studies of the protonation constants and Cu2+ complex stability constants of pituitary adenylate cyclase activating polypeptide fragments (HSDGI-NH2, TDSYS-NH2, RKQMAVKKYLAAVL-NH2). With HSDGI-NH2, the formation of a dimeric complex Cu2H-2L2 was found in the pH range 5-8, in which the coordination of copper(II) is glycylglycine-like, while the fourth coordination site is occupied by the imidazole N3 nitrogen atom, forming a bridge between two copper(II) ions. The formation of dimeric species does not prevent the deprotonation and coordination of the amide nitrogen, and in pH above 8 the CuH-2L complex is formed. Aspartic acid in the third position of peptide sequence stabilizes the CuH-2L species and prevents the coordination of a fourth nitrogen donor. Aspartic acid residue in the second position of TDSYS-NH2 stabilizes the CuL (2N) complex but does not prevent deprotonation and binding of the second and third peptide nitrogens to give 3N and 4N complexes at higher pH. The tetradecapeptide amide forms with copper(II) ions unusually stable 3N and 4N complexes compared to pentaalanine amide.     

Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes

Pituitary adenylate cyclase activating polypeptide (PACAP) peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway, including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC(1)null, PAC(1)HOP1, and VPAC(2) receptors; the urothelium was more restricted in expressing preferentially the PAC(1) receptor subtype only. Immunocytochemical studies for PAC(1) receptors were consistent with these tissue distributions. Furthermore, the addition of 50-100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 muM) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, vasoactive intestinal polypeptide had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC(1) receptor antagonist, PACAP6-38, reduced cystitis-induced bladder overactivity. In summary, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis.     

Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline.

Pituitary adenylate cyclase activating polypeptide (PACAP) provoked the rat chromaffin cells to secrete adrenaline. Within 20 min, the amount of adrenaline secreted by PACAP (10(-8) M) was as much as that caused by acetylcholine (10(-4) M). PACAP, but not acetylcholine, induced a long-term (over 120 min) increase in secretion of adrenaline. PACAP also activated adenylate cyclase and elevated cytosolic Ca2+ concentration. Furthermore, we found immunoreactive PACAP and PACAP binding sites in the rat adrenal medulla. These results suggest that PACAP has an important role in stimulating secretion of adrenaline in the adrenal medulla.     

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.     

垂体腺苷酸环化酶激活肽的发现及研究进展

垂体腺苷酸环化酶激活肽是最初在绵羊下丘脑发现的一种新的具有多种生物活性的多肽。它广泛分布于中枢神经系统、周围神经系统以及非神经组织内。此外,它在某些类型细胞的旁分泌和自分泌主财节中也发挥作用。     

Investigation of pituitary adenylate cyclase activating polypeptide (PACAP) in human amniotic fluid samples

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide acting as a hormone, a neuromodulator, a neurotransmitter, a trophic factor and is involved in a variety of developmental and regenerative processes. PACAP is present in several human tissues and biological fluids. In many pathological conditions, changes in PACAP levels have been described to reflect disease progression, therefore PACAP has diagnostic value as a potential biomarker. Since PACAP has been shown to play an important role in reproductive physiology and development, it was of interest to examine whether this neuropeptide occurs in the human amniotic fluid. Amniotic fluid samples were collected between the 15-19^th weeks of gestation from volunteering pregnant women undergoing amniocentesis as a prenatal diagnostic tool due to maternal age. Pathological cases were excluded after prenatal karyotype analysis. PACAP-like immunoreactivity was measured by radioimmunoassay and could be detected in all samples. The present study provides evidence for the presence of PACAP in human amniotic fluid, but determination of the exact physiological or pathological significance awaits further investigation.     

Hyperpolarization-activated Cl current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1–10 μM) activated an inward rectifier current at membrane potentials more negative than −60 mV without causing any significant change in currents at potentials more positive than −60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at −22.5 mV, close to the theoretical value of Cl⁻ equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl⁻]_o. This current was blocked by Cl⁻ channel blocker SITS and Ba²⁺. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl⁻ current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

Accumulation of unique globo-series glycolipids in PC 12h pheochromocytoma cells

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.     

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.     

斜带石斑鱼PACAP的原核表达及活性分析

自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽（Pituitary adenylate cyclase activating polypeptide,PACAP）以来（Miyata et al．,1989）,已证明它能促进垂体激素释放,同时还具有神经递质、神经调质和神经营养等作用,使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽（VIP）-胰高血糖素-生长激素释放因子-分泌素家族（Campbell and Scanes,1992）成员,已鉴别出包含27和38个氨基酸两种类型。对原索动物（McRory et al．,1997）、两栖类（蛙）（Alexandre et al．,2000）、爬行类（蜥蜴）（Pohland Wank,1998）、鸟类（鸡）（McRory et al．,1997）,啮齿类（鼠）（Ghatei et al．,1993）等脊椎动物PACAP的研究多集中在结构与进化方面,对功能了解甚少。