Peptides (v.28, #9)

Contents (VII).

Announcement (VI).

Editorial by Hubert Vaudry (1615-1616).

PACAP: The road to discovery by Akira Arimura (1617-1619).

Novel analogs of VIP with multiple C-terminal domains by David Dangoor; Sara Rubinraut; Mati Fridkin; Illana Gozes (1622-1630).
The effect of multiplication of the N-terminal domain of vasoactive intestinal peptide (VIP) on the binding activity of the peptide was recently evaluated. A VIP analog with multiple N-terminal domains was found to be slightly more potent as compared to [Nle17]VIP towards VIP receptor type 1 (VPAC1)—related cAMP production. Here, the effect of multiplication of the C-terminal domain of VIP was evaluated with the aim of possibly amplifying peptide-receptor (VPAC1) binding and activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the C-terminal domain that was obtained by either a simple linear tandem extension or by a unique branching methodology. Results show that despite significant alterations in the C-terminal domain of VIP that is considered essential to induce potent receptor binding, few peptides demonstrated only slight reduction in receptor binding and activation in comparison to [Nle17]VIP. Furthermore, a specific branched VIP analog with multiple C-terminal domains was equipotent to [Nle17]VIP in the cAMP production assay. Therefore, it is concluded that the association between the VIP ligand to the VIP receptor could be tolerable to size increases in the C-terminal region of the VIP ligand and multiplication of the C-terminal does not increase activity.
Keywords: VIP; VPAC1; cAMP; HT-29 cells; Peptides; Receptor binding;

VIP and PACAP impact strongly on human pathophysiology. Their receptors are very promising targets for developing new drugs in the treatment of inflammatory and neurodegenerative diseases. This article reviews the present knowledge regarding VIP and PACAP receptors, i.e. VPAC1, VPAC2 and PAC1. This includes: (I) a critical review of instrumental peptide agonists and antagonists; (II) a survey of recent data regarding the structure of VPAC1 receptor and the docking of VIP in the receptor binding domain. Structural models for the VPAC2 and PAC1 receptor N-terminal ectodomains are also described; (III) A critical description of the two models of VPAC1 receptor activation in the general context of class II/family B G protein-coupled receptors.
Keywords: VPAC receptor; PAC1 receptor; G protein-coupled receptor; Class II/family B; N-terminal ectodomain; Peptide agonist;

Vasoactive intestinal peptide (VIP) is one of the major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. In an airway system where VIP-immunoreactive nerve fibers are present, VIP acts as neurotransmitter or neuromodulator of the inhibitory non-adrenergic and non-cholinergic airway nervous system and influences many aspects of pulmonary biology. A clinical application of VIP has been believed to offer potential benefits in the treatment of chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, its clinical application has been limited in the past for a number of reasons, including its extremely short plasma half-life after intravenous administration and difficulty in administration routes. The development of long-acting VIP analogues, in combination with appropriate drug delivery systems, may provide clinically useful agents for the treatment of asthma/COPD. In this review, development of efficacious VIP derivatives, drug delivery systems designed for VIPs and the potential application for asthma/COPD are discussed. We also include original data from our chemical modification experiments and formulation studies, which led to successful development of [R15, 20, 21, L17]-VIP-GRR (IK312532), a potent VIP analogue, and a VIPs-based dry powder inhaler system.
Keywords: VIP; Asthma; COPD; Dry powder inhaler; Drug delivery system;

Maxadilan, a PAC1 receptor agonist from sand flies by Ethan A. Lerner; Aurel O. Iuga; Vemuri B. Reddy (1651-1654).
In 1991, a potent 61 amino acid vasodilator peptide, named maxadilan, was isolated from the salivary glands of the sand fly. Subsequently, it was shown that this peptide specifically and potently activated the mammalian PAC1 receptor, one of the three receptors for PACAP. These studies and the link between maxadilan and leishmaniasis are discussed.
Keywords: Maxadilan; PAC1; Receptor; PACAP; Leishmaniasis;

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: An update by Jean-Marc Muller; Colin Debaigt; Stéphanie Goursaud; Alicia Montoni; Nicolas Pineau; Annie-Claire Meunier; Thierry Janet (1655-1666).
The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.
Keywords: VIP and related peptides receptors; PHI and PHM binding sites; GTP insensitive binding sites; Accessory proteins; Receptor activity modifying proteins (RAMP);

Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells by Cha Xiang Guan; Yan Ru Cui; Min Zhang; Hong Bo Bai; Ravitej Khunkhun; Xiang Fang (1667-1673).
Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays an important role in maintaining the bronchial tone of the airway and has anti-inflammatory properties. Recently, we reported that VIP enhances wound repair in human bronchial epithelial cells (HBEC). In the present study, we have identified the intracellular signaling molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC. Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and mRNA induced by VIP. These results suggest that intracellular signaling molecules such as PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.
Keywords: Vasoactive intestinal peptide; Human bronchial epithelial cells; Wound healing; PKA; PKC; Calmodulin; ERK;

Methamphetamine-induced hyperactivity and behavioral sensitization in PACAP deficient mice by Harumi Fujii; Toshihiro Ishihama; Yukio Ago; Norihito Shintani; Michiya Kakuda; Hitoshi Hashimoto; Akemichi Baba; Toshio Matsuda (1674-1679).
Mice lacking the PACAP gene (PACAP−/−) display psychomotor abnormalities such as novelty-induced hyperactivity and jumping behavior, and they show different responses to amphetamine, a typical psychostimulant. The present study examined the possible role of endogenous PACAP in methamphetamine (METH)-induced hyperactivity and behavioral sensitization. The locomotor activity of hyperactive PACAP−/− mice was measured using the infrared photocell beam detection system, Acti-Track, after a habituation period. Single administration of METH (1 and 2 mg/kg) caused a robust increase in locomotor activity of mice, but this effect did not differ between wild-type and PACAP−/− mice. Repeated administration of METH (1 mg/kg) for 7 days enhanced METH-induced hyperactivity, and this sensitization was observed even when withdrawn for 7 days. There was no difference in the degree of development and expression of METH-induced behavioral sensitization between wild-type and PACAP−/− mice. In addition, there was no difference in METH-induced increases in extracellular serotonin and dopamine levels in the prefrontal cortex of the normal and sensitized mice between the two groups. These results suggest that endogenous PACAP is not involved in the locomotor stimulant activity of acute METH and repeated METH-induced behavioral and neurochemical sensitization.
Keywords: PACAP−/− mice; PACAP; Methamphetamine (METH); Locomotor activity; Behavioral sensitization;

Knocked down and out: PACAP in development, reproduction and feeding by Nancy M. Sherwood; Bruce A. Adams; Emma R. Isaac; Sheng Wu; Erica A. Fradinger (1680-1687).
One approach to understanding the role of PACAP in vivo is to knockdown the translation of PACAP mRNA to protein or to knock out the PACAP gene by targeted disruption. In this paper, we review the effect of PACAP knockdown with morpholinos on early brain development in zebrafish. Also reviewed is the role of PACAP at several stages of reproduction as assessed in mice with a disrupted PACAP gene. New data are presented to analyze PACAP's action in energy homeostasis (body mass, food intake, endocrine parameters) using female PACAP-null mice. The evidence suggests PACAP is important for brain development in zebrafish and is required for normal reproduction, but not for body mass or food intake in mice maintained near thermoneutrality.
Keywords: Morpholino; PACAP gene knockout; Brain development; Reproduction; Energy balance; High fat diet; Mice; Zebrafish;

Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: A model for the study of intestinal ileus and Hirschsprung's disease by V. Lelievre; G. Favrais; C. Abad; H. Adle-Biassette; Y. Lu; P.M. Germano; G. Cheung-Lau; J.R. Pisegna; P. Gressens; G. Lawson; J.A. Waschek (1688-1699).
In 1970, Drs. Said and Mutt isolated a novel peptide from porcine intestinal extracts with powerful vasoactive properties, and named it vasoactive intestinal peptide (VIP). Since then, the biological actions of VIP in the gut as well as its signal transduction pathways have been extensively studied. A variety of in vitro and in vivo studies have indicated that VIP, expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons, is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP actions are believed to be mediated mainly by interactions with highly expressed VPAC1 receptors and the production of nitric oxide (NO). Furthermore, VIP has been implicated in numerous physiopathological conditions affecting the human gut, including pancreatic endocrine tumors secreting VIP (VIPomas), insulin-dependent diabetes, Hirschsprung's disease, and inflammatory bowel syndromes such as Crohn's disease and ulcerative colitis. To further understand the physiological roles of VIP on the GI tract, we have begun to analyze the anatomical and physiological phenotype of C57BL/6 mice lacking the VIP gene. Herein, we demonstrate that the overall intestinal morphology and light microscopic structure is significantly altered in VIP−/− mice. Macroscopically there is an overall increase in weight, and decrease in length of the bowel compared to wild type (WT) controls. Microscopically, the phenotype was characterized by thickening of smooth muscle layers, increased villi length, and higher abundance of goblet cells. Alcian blue staining indicated that the latter cells were deficient in mucus secretion in VIP−/− mice. The differences became more pronounced from the duodenum to the distal jejunum or ileum of the small bowel but, became much less apparent or absent in the colon with the exception of mucus secretion defects. Further examination of the small intestine revealed larger axonal trunks and unusual unstained patches in myenteric plexus. Physiologically, the VIP−/− mice showed an impairment in intestinal transit. Moreover, unlike WT C57BL/6 mice, a significant percentage of VIP−/− mice died in the first postnatal year with overt stenosis of the gut.
Keywords: Vasoactive intestinal peptide; Knockout; Peristalsis; Gastrointestinal transit;

In neurons from rat hippocampus, VIP induces the elongation of dendrites. In the present study, we have investigated in cultured hippocampal neurons whether VIP changed the actin and tubulin cytoskeleton in dendrites. VIP caused the elongation of dendrites and induced the outgrowth of microtubules, so that they extended up to the tips. In contrast, VIP reduced the F-actin content measured as total pixel after phalloidin staining in dendritic tips. These results suggest that VIP causes dendrite elongation by facilitating the outgrowth of microtubules into the newly formed extensions.
Keywords: Dendrite; Elongation; F-actin; Tubulin; VIP; RhoA;

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) concentration (0.001–1000 nM)-dependently stimulated cyclic AMP production in rat primary neuronal and glial cell (astrocyte) cultures. The actions of both peptides were much more pronounced in astrocytes than in neuronal cultures. Stimulatory effects of PACAP and VIP on cyclic AMP formation were significantly smaller in cell cultures subjected to 24 h lasting hypoxic conditions, induced either chemically (100 μM cobalt chloride) or by low 3% oxygen hypoxia, compared to the normoxic condition (95% air and 5% CO2). This picture contrasted with the effects of forskolin that were similar under normoxic and hypoxic conditions. It is suggested that hypoxia leads to changes in PACAP- and VIP-driven cyclic AMP-dependent signaling in the rat brain by influencing molecular processes likely occurring at the level of receptor protein or receptor-Gs protein coupling.
Keywords: Pituitary adenylate cyclase activating polypeptide (PACAP); Vasoactive intestinal peptide (VIP); Cyclic AMP; Neurons; Astrocytes; Rat; Hypoxia;

Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development by Jun Watanabe; Tomoya Nakamachi; Ryousuke Matsuno; Daisuke Hayashi; Masahisa Nakamura; Sakae Kikuyama; Shigeo Nakajo; Seiji Shioda (1713-1719).
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.
Keywords: Pituitary adenylate cyclase-activating Polypeptide (PACAP); PAC1 receptor; Neural development; Neurogenesis;

The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.
Keywords: Excitotoxicity; Astrocytes; Chemokines; MAPK; cAMP; Calcium; Growth factors; Second messengers; Secretion; Receptors; gp120;

Involvement of VIP and PACAP in neonatal brain lesions generated by a combined excitotoxic/inflammatory challenge by Geraldine Favrais; Alain Couvineau; Marc Laburthe; Pierre Gressens; Vincent Lelievre (1727-1737).
Several reports have highlighted the potential roles for the VIP-related neuropeptides in regeneration/neuroprotection after brain or nerve injuries. We previously reported that peripheral inflammation worsened ibotenate-induced cystic white matter lesions. Because VIP is also known as an immunomodulator, we wonder if VIP could also limit the deleterious effects of local inflammation. Therefore, we first tested the effects of peripheral IL-1β on VIP and PACAP central production. Second, we observed that cox-2 activation by IL-1β was essential to generate changes in ligand/receptor gene expression. We further tested whether the intraperitoneal injection of IL-1β, known to aggravate the ibotenate-induced lesions, could modify the expression pattern of VIP-related genes. Finally, we concluded using histological analysis that VIP[ala11,22,28], a synthetic VPAC1 agonist completely reversed the aggravating effects of IL-1β on ibotenate-induced lesions of the periventricular white matter. Conversely, VIP-neurotensin hybrid, a nonselective VIP receptor antagonist, worsened the lesions. All together, our results suggest that an activation of VIP/VPAC1 signaling cascade in the vicinity of the injury site could circumvent the synergizing degenerative effects of ibotenate and pro-inflammatory cytokines. Therefore, development of therapeutic tools inducing/sustaining the activation of VIP/VPAC1 signaling cascade may lead to future preventive treatments for inflammatory conditions during pregnancy.
Keywords: VIP signaling; Pro-inflammatory cytokines; Brain excitoxicity; Ibotenate; White matter degeneration; Cyclooxygenase;

Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta by Monica Ciarlo; Federica Bruzzone; Cristiano Angelini; Mauro Vallarino; Hubert Vaudry (1738-1745).
The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.
Keywords: PACAP receptors; Frog development; Tissue expression; In situ hybridization;

Neurotrophic effects of PACAP in the cerebellar cortex by Béatrice Botia; Magali Basille; Aurélie Allais; Emilie Raoult; Anthony Falluel-Morel; Ludovic Galas; Valérie Jolivel; Olivier Wurtz; Hitoshi Komuro; Alain Fournier; Hubert Vaudry; Delphine Burel; Bruno J. Gonzalez; David Vaudry (1746-1752).
In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.
Keywords: Cerebellar granule neurons; PACAP; cAMP/PKA; Apoptosis; Caspase-3; Neuroprotection;

Role of PACAP and VIP in astroglial functions by Olfa Masmoudi-Kouki; Pierrick Gandolfo; Hélène Castel; Jérôme Leprince; Alain Fournier; Agnieszka Dejda; Hubert Vaudry; Marie-Christine Tonon (1753-1760).
Astrocytes represent at least 50% of the volume of the human brain. Besides their roles in various supportive functions, astrocytes are involved in the regulation of stem cell proliferation, synaptic plasticity and neuroprotection. Astrocytes also influence neuronal physiology by responding to neurotransmitters and neuropeptides and by releasing regulatory factors termed gliotransmitters. In particular, astrocytes express the PACAP-specific receptor PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R during development and/or in the adult. There is now clear evidence that PACAP and VIP modulate a number of astrocyte activities such as proliferation, plasticity, glycogen production, and biosynthesis of neurotrophic factors and gliotransmitters.
Keywords: Astrocytes; Stem cell; Plasticity; Neurotrophic factors; Gliotransmitters;

The hypothalamic region of the brain in vertebrates is a center that plays an important role in feeding regulation. Many kinds of hypothalamic neuropeptides or peripheral transmitters, such as orexin, neuropeptide Y, Agouti-related peptide, melanin-concentrating hormone, proopiomelanocortin-derived peptides, galanin, galanin-like peptide, ghrelin, corticotropin releasing hormone, cholecystokinin, cocaine amphetamine-related transcript peptides and leptin, have been implicated in the regulation of feeding behavior, psychomotor activity and energy homeostasis in rodents. Recent studies have also examined the effects of these neuropeptides or factors on food intake in non-mammalian vertebrates, especially chick and goldfish, and the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) in feeding behavior, locomotor activity or psychomotor activity in vertebrates. This article gives an overview of the regulation of feeding behavior and related physiology by PACAP and VIP in vertebrates in order to clarify the appetite-regulating system mediated by the two peptides.
Keywords: Feeding regulation; PACAP; VIP; Vertebrates;

Several features of the molecular circadian oscillator of the chicken pineal gland show homology with those in the mammalian SCN. Studies have shown the effects of PACAP on the mammalian SCN, but its effects on the expression of clock genes in the avian pineal gland have not yet been demonstrated. Clock and Cry1 expression was analyzed in pineal glands of chicken embryos after exposure to PACAP-38 in vitro. PACAP reduced expression of both clock genes within 2 h. Ten hours after exposure, mRNA contents exceeded that of the controls. Our results support the hypothesis that the molecular clock machinery in the chicken pineal gland is also sensitive to PACAP.
Keywords: Circadian; Avian; Pineal gland; PACAP; Clock; Cry1; Light;

PACAP and PDF signaling in the regulation of mammalian and insect circadian rhythms by Inge Mertens; Steven J. Husson; Tom Janssen; Marleen Lindemans; Liliane Schoofs (1775-1783).
Endogenous circadian clocks are inherent to all living organisms. They are needed to guarantee successful life since they regulate very important biological processes such as behavior and reproduction. Secretin-like G-protein coupled receptors are very important factors in the signal transduction pathways of circadian clocks. In this review, we will focus on the role of two secretin-like signaling pathways that play an important role in the regulation of the mammalian and the insect clock, respectively: the pituitary adenylate cyclase-activating polypeptide (PACAP) and pigment dispersing factor (PDF) signaling pathways. Both pathways are most likely related although their function in the biological clock differs.
Keywords: PACAP; PACAP-receptor; PDF; PDF-receptor; Circadian rhythms; Amnesiac; Drosophila;

VIP and PACAP stimulate TSH release from the bullfrog pituitary by Reiko Okada; Kazutoshi Yamamoto; Yoichi Ito; Hiroshi Mochida; Marie-Christine Tonon; Alain Fournier; Jérôme Leprince; Hubert Vaudry; Sakae Kikuyama (1784-1789).
We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin (TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal polypeptide (VIP) (fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24 h with graded concentrations of fVIP, fPACAP38 and PACAP27 (10−9 to 10−6  M) induced a dose-dependent stimulation of TSH release with minimum effective doses of 10−9  M for fVIP and 10−8  M for fPACAP38 and PACAP27. The PAC1-R/VPAC2-R antagonist PACAP6–38 (10−7 and 10−6  M) dose-dependently suppressed the stimulatory effects of fVIP and fPACAP38 (10−7  M each). Likewise, this antagonist (10−6 and 10−5  M) dose-dependently attenuated the stimulatory effect of PACAP27 (10−7  M). On the other hand, the VPAC1-R/VPAC2-R antagonist [d-pCl-Phe6, Leu17]VIP (10−6 and 10−5  M) dose-dependently inhibited the stimulatory effect of fVIP (10−9  M) and PACAP27 (10−8  M), but did not affect the response to fPACAP38 (10−8  M). These data indicate that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R.
Keywords: VIP; PACAP; Frog TSH; VPAC2-receptor; PAC1-receptor;

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis by Adhanet H. Kidane; Peter M.J.M. Cruijsen; Maria A. Ortiz-Bazan; Hubert Vaudry; Jerome Leprince; Frouwke J. Kuijpers-Kwant; Eric W. Roubos; Bruce G. Jenks (1790-1796).
The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.
Keywords: PACAP; VIP; α-MSH; Pars nervosa; Pars intermedia; Melanotrope cell;

What is the role of PACAP in gonadotrope function? by Raymond Counis; Jean-Noël Laverrière; Ghislaine Garrel-Lazayres; Joëlle Cohen-Tannoudji; Sigolène Larivière; Christian Bleux; Solange Magre (1797-1804).
Strong evidence in favor of a direct action of hypothalamic PACAP at the pituitary to modulate gonadotrope function has been acquired mainly by in vitro studies using cultured pituitary cells or gonadotrope cell lines. In particular, PACAP has been shown to cooperate with GnRH, the primary regulator of gonadotropes, to regulate/modulate gonadotropin subunit gene expression, gonadotropin release as well as gonadotrope responsiveness. These effects of PACAP appear to be due essentially to its high potent stimulatory action on the cAMP/protein kinase pathway. Ensuing mechanisms include signaling cross-talk and/or enhanced gene expression within gonadotropes. PACAP may also indirectly operate on these cells through paracrine mechanisms. While PACAP has long been viewed as a hypophysiotropic factor, a locally produced PACAP has also been described. Interestingly, both appear similarly up-regulated at proestrus of the reproductive cycle in female rats. Further in vivo investigation is now necessary to ascertain the physiological relevance of the observed pituitary PACAP effects and especially to evaluate the respective contribution of hypothalamic and pituitary PACAP in the dynamic control of gonadotrope function.
Keywords: PACAP; GnRH; Pituitary; Gonadotrope cells; Signaling pathways; Cross-talk;

Role of VIP and PACAP in islet function by Maria Sörhede Winzell; Bo Ahrén (1805-1813).
Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two closely related neuropeptides that are expressed in islets and in islet parasympathetic nerves. Both peptides bind to their common G-protein-coupled receptors, VPAC1 and VPAC2, and PACAP, in addition to the specific receptor PAC1, all three of which are expressed in islets. VIP and PACAP stimulate insulin secretion in a glucose-dependent manner and they both also stimulate glucagon secretion. This action is achieved through increased formation of cAMP after activation of adenylate cyclase and stimulation of extracellular calcium uptake. Deletion of PAC1 receptors or VPAC2 receptors results in glucose intolerance. These peptides may be of importance in mediating prandial insulin secretion and the glucagon response to hypoglycemia. Animal studies have also suggested that activation of the receptors, in particular VPAC2 receptors, may be used as a therapeutic approach for the treatment of type 2 diabetes. This review summarizes the current knowledge of the potential role of VIP and PACAP in islet function.
Keywords: PACAP; VIP; Islets; Insulin secretion; Receptor; Parasympathetic;

A novel VIP signaling pathway in T cells by Li Liu; Jui-Hung Yen; Doina Ganea (1814-1824).
Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory agent. In addition to the deactivation of macrophages, dendritic cells, and microglia, VIP shifts the Th1/Th2 balance, promoting the preferential differentiation and survival of Th2 cells, to the detriment of the proinflammatory Th1 effectors. Several mechanisms operate in the Th1/Th2 shift induced by VIP. Here we report on a novel mechanism for the effect of VIP on T cell differentiation, and show that VIP inhibits Th1 differentiation by interfering directly with the IL-12 Jak2/STAT4 signaling pathway in T cells. The effect of VIP is cAMP-dependent, and appears to be mediated through the activation of protein tyrosine phosphatases (PTP), with SHP-2 as a potential target. The activation of PTPs represents a novel cAMP-downstream target for the immunomodulatory effects of VIP.
Keywords: Vasoactive intestinal peptide; Th1 differentiation; Jak2/STAT4 signaling pathway; Protein tyrosine phosphatases (PTP); SHP-2 protein tyrosine phosphatase;

Regulation of TLR expression, a new perspective for the role of VIP in immunity by R.P. Gomariz; A. Arranz; Y. Juarranz; I. Gutierrez-Cañas; M. Garcia-Gomez; J. Leceta; C. Martínez (1825-1832).
The contribution of VIP immune functions to the regulation of homeostasis and health is well known. Modulation of immune responses through new therapeutics is one of the main goals of physicians and scientists seeking to control inflammatory/autoimmune diseases in humans. Initial therapeutic strategies targeted adaptive immune responses; discovery of Toll-like receptors (TLR) has widened the horizon to include targeting the innate immune system. In this review we have summarized recent information about VIP modulation of TLR function, and we suggest that VIP represents a new therapeutic option in the management of several pathologies.
Keywords: Neuroimmunomodulation; Vasoactive intestinal peptide; Toll-like receptors;

Tuning immune tolerance with vasoactive intestinal peptide: A new therapeutic approach for immune disorders by David Pozo; Elena Gonzalez-Rey; Alejo Chorny; Per Anderson; Nieves Varela; Mario Delgado (1833-1846).
The induction of immune tolerance is essential for the maintenance of immune homeostasis and to limit the occurrence of exacerbated inflammatory and autoimmune conditions. Multiple mechanisms act together to ensure self-tolerance, including central clonal deletion, cytokine deviation and induction of regulatory T cells. Identifying the factors that regulate these processes is crucial for the development of new therapies of autoimmune diseases and transplantation. The vasoactive intestinal peptide (VIP) is a well-characterized endogenous anti-inflammatory neuropeptide with therapeutic potential for a variety of immune disorders. Here, we examine the latest research findings, which indicate that VIP participates in maintaining immune tolerance in two distinct ways: by regulating the balance between pro-inflammatory and anti-inflammatory factors, and by inducing the emergence of regulatory T cells with suppressive activity against autoreactive T-cell effectors.
Keywords: Inflammation; Autoimmunity; Regulatory T cells; Tolerance; Neuroimmunology; Neuropeptide;

Inhibitory effect of PACAP-38 on acute neurogenic and non-neurogenic inflammatory processes in the rat by Z. Helyes; G. Pozsgai; R. Börzsei; J. Németh; T. Bagoly; L. Márk; E. Pintér; G. Tóth; K. Elekes; J. Szolcsányi; D. Reglődi (1847-1855).
Inhibitory actions of pituitary adenylate cyclase activating polypeptide (PACAP) have been described on cellular/vascular inflammatory components, but there are few data concerning its role in neurogenic inflammation. In this study we measured PACAP-like immunoreactivity with radioimmunoassay in the rat plasma and showed a two-fold elevation in response to systemic stimulation of capsaicin-sensitive sensory nerves by resiniferatoxin, but not after local excitation of cutaneous afferents. Neurogenic plasma extravasation in the plantar skin induced by intraplantar capsaicin or resiniferatoxin, as well as carrageenan-induced paw edema were significantly diminished by intraperitoneal PACAP-38. In summary, these results demonstrate that PACAP is released from activated capsaicin-sensitive afferents into the systemic circulation. It diminishes acute pure neurogenic and mixed-type inflammatory reactions via inhibiting pro-inflammatory mediator release and/or by acting at post-junctional targets on the vascular endothelium.
Keywords: Capsaicin-sensitive sensory nerves; Resiniferatoxin; Sciatic nerve stimulation; Carrageenan-induced edema; Plasma PACAP-like immunoreactivity; MALDI TOF;

Microarray analyses of pituitary adenylate cyclase activating polypeptide (PACAP)-regulated gene targets in sympathetic neurons by Karen M. Braas; Kristin C. Schutz; Jeffrey P. Bond; Margaret A. Vizzard; Beatrice M. Girard; Victor May (1856-1870).
The high and preferential expression of the PAC1(short)HOP1 receptor in postganglionic sympathetic neurons facilitates microarray studies for mechanisms underlying PACAP-mediate neurotrophic signaling in a physiological context. Replicate primary sympathetic neuronal cultures were treated with 100 nM PACAP27 either acutely (9 h) or chronically (96 h) before RNA extraction and preparation for Affymetrix microarray analysis. Compared to untreated control cultures, acute PACAP treatment modulated significantly the expression of 147 transcripts of diverse functional groups, including peptides, growth factors/cytokines, transcriptional factors, receptors/signaling effectors and cell cycle regulators, that collectively appeared to facilitate neuronal plasticity, differentiation and/or regeneration processes. Some regulated transcripts, for example, were related to BDNF/TrkB, IL-6/Jak2/Socs2 and TGF/follistatin signaling; many transcripts affected bioactive peptide and polyamine biosynthesis. Although chronic PACAP treatments altered the expression of 109 sympathetic transcripts, only 43 transcripts were shared between the acute and chronic treatment data sets. The PACAP-mediated changes in transcript expression were corroborated independently by quantitative PCR measurement. The PACAP-regulated transcripts in sympathetic neurons did not bear strong resemblance to those in PACAP-treated pheochromocytoma cells. However, many PACAP-targeted sympathetic transcripts, especially those related to peptide plasticity and nerve regeneration processes, coincided significantly with genes altered after peripheral nerve injury. The ability for sympathetic PAC1(short)HOP1 receptors to engage multiple downstream signaling cascades appeared to be reflected in the number and diversity of genes targeted in a multifaceted strategy for comprehensive neurotrophic responses.
Keywords: PACAP; PAC1 receptor; Sympathetic; Superior cervical ganglion; Neurotrophic;

Meta-analysis of microarray-derived data from PACAP-deficient adrenal gland in vivo and PACAP-treated chromaffin cells identifies distinct classes of PACAP-regulated genes by Babru Samal; Matthew J. Gerdin; David Huddleston; Chang-Mei Hsu; Abdel G. Elkahloun; Nikolas Stroth; Carol Hamelink; Lee E. Eiden (1871-1882).
Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes also regulated by either TNFα or TGFβ, two first messengers acting on transcription mainly through NFκB and Smads, respectively.
Keywords: Adrenal gland; Bovine chromaffin cells; Btg2; C/EBP; Ier3; Meta-analysis; Microarray; Middle cerebral artery occlusion; Neuroprotection; NFκB; PACAP; PC12; Scmh1; Secretogranin; Smad; TGFβ; TNFα; YWAHZ;

Vasoactive intestinal peptide–camptothecin conjugates inhibit the proliferation of breast cancer cells by Terry W. Moody; Samuel A. Mantey; Joseph A. Fuselier; David H. Coy; Robert T. Jensen (1883-1890).
The effects of vasoactive intestinal peptide–camptothecin (VIP–CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala2,8,9,19,24.25.27, Nle17, Lys28)VIP, (A-NL-K)VIP, was synthesized and Lys28 was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC1-R, with IC50 values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of 125I-VIP to 3T3 cells transfected with VPAC1-R with IC50 values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of 125I-Ro25-1553 to 3T3 cells transfected with VPAC2-R with IC50 values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. 125I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 °C but not 4 °C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC1-R agonist which is cytotoxic for breast cancer cells.
Keywords: Camptothecin; VIP receptors; Cellular proliferation; Breast cancer; Receptor-mediated cytotoxicity;

We have recently shown significant renoprotective effects with the administration of pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney. PACAP markedly inhibited the production of proinflammatory cytokines stimulated by immunoglobulin light chains in human renal proximal tubule epithelial cells and in the kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of 4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood rapidly declined after the cessation of administration with a half-life of about 5–10 min. The continuous infusion did not significantly alter the basal glucose level, blood gases or blood pressure. There was a large reduction in free lambda light chains in urine after the start of the treatment with PACAP. These studies show that PACAP can be safely used in humans and suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney.
Keywords: Cytokines; Inflammation; Light-chain immunoglobulins; Mitogen-activated protein kinase; Myeloma kidney; PACAP; PACAP receptors;

Vasoactive intestinal peptide enhances growth and angiogenesis of human experimental prostate cancer in a xenograft model by Beatriz Collado; María J. Carmena; Celia Clemente; Juan C. Prieto; Ana M. Bajo (1896-1901).
We show that vasoactive intestinal peptide (VIP) exerts trophic and proangiogenic activities in experimental prostate cancer in vivo. Nude mice were subcutaneously injected with Matrigel impregnated with LNCaP prostate cancer cells. Cell treatment with 100 nM VIP for 1 h before xenograft resulted in increased tumor growth after 8 and, more remarkably, 15 days of injection. The same occurred with the mRNA expression of the main angiogenic factor, vascular endothelial growth factor (VEGF), as shown by real-time RT-PCR quantification. The proangiogenic activity of VIP was further established by showing increases of hemoglobin levels, Masson trichromic staining, and immunohistochemical CD34 staining in tumors excised 15 days after subcutaneous injection of VIP-treated cells as compared to control conditions. All these parameters indicate that VIP increases vessel formation. This xenograft model is a useful tool to study in vivo the effects of VIP-related peptides in tumor growth and development of blood supply as well as their therapeutical potential in prostate cancer.
Keywords: VIP; Tumor promotion; Xenograft; Angiogenesis; VEGF; Prostate cancer;

PACAP, VIP and their receptors in the metazoa: Insights about the origin and evolution of the ligand–receptor pair by João C.R. Cardoso; Florbela A. Vieira; Ana S. Gomes; Deborah M. Power (1902-1919).
The evolution, function and interaction of ligand–receptor pairs are of major pharmaceutical interest. Comparative sequence analysis approaches using data from phylogenetically distant organisms can provide insights into their origin and possible physiological roles. The present review focuses on the pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and their receptors in the metazoa. A PACAP-like peptide is present in tunicates and chordates while VIP- and PACAP/VIP-specific receptors have only been isolated in the latter phyla. The apparently disparate evolution of the ligands and their specific receptors raises questions about their evolution during the metazoan radiation and also about how the ligands may have acquired new functions.
Keywords: Evolution; GPCRs; Ligand–receptor; Metazoan; PACAP; VIP;

PACAP-related peptide (PRP)—Molecular evolution and potential functions by Janice K.V. Tam; Leo T.O. Lee; Billy K.C. Chow (1920-1929).
PACAP-related peptide (PRP) and PACAP are structurally related peptides that are encoded in the same transcripts. In the past, it was believed that the mammalian PRPs are evolved from GHRHs in non-mammals. With the recent discovery of authentic GHRH and receptor genes in frog and fish, this review aims to (1) coin the name of all GHRH-like peptides in previous literature as PRPs and (2) provide the background for new research direction for PRP in vertebrates. As a goldfish receptor highly specific for PRP with distinct tissue distribution has previously been characterized, it is highly possible that PRP plays a physiological role in non-mammalian vertebrates and the function of PRP has somehow been lost in mammals as a consequence of the loss of its receptor in the genome. This information may provide clues to elucidate functions of PRP in the future.
Keywords: Pituitary adenylate cyclase-activating polypeptide; PACAP-related peptide; Evolution; Growth hormone release; Growth hormone-releasing hormone; Growth hormone-releasing hormone-like peptide;