The 6th International Symposium on VIP, PACAP and Related Peptides

Regulatory Peptides 115 (2003) 37 – 71 www.elsevier.com/locate/regpep

Abstracts (in alphabetical order after first author)

The 6th International Symposium on VIP, PACAP and Related Peptides Hakone, Japan September 1–4, 2003

Chairman: Seiji Shioda Department of Anatomy Showa University School of Medicine Tokyo, Japan

doi:10.1016/S0167-0115(03)00166-6

Abstracts

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The 6th International Symposium on VIP, PACAP and Related Peptides

Therapeutic effects of VIP in the TNBS mice model of Crohn’s disease C. Abad, C. Martinez, M.G. Juarranz, A. Arranz, J. Leceta, M. Delgado and R.P. Gomariz Faculty of Biology, Department of Cell Biology, Complutense University, Madrid, Spain Crohn’s disease (CD) is an inflammatory chronic pathology of the gut, which courses with severe diarrhea, weight loss and pain. TNBS administration in mice resembles the main symptoms and molecular features of CD. As VIP has a beneficial effect in both inflammatory (septic shock) and Th1 diseases (rheumatoid arthritis), we investigated the effect of VIP on the TNBS colitis model in mice. VIP reduced diarrhea and weight loss of TNBS treated mice, reestablishing the normal macroscopic and microscopic aspect of the colon. VIP was effective even if given when the disease was already stablished. The action of VIP was mediated through the inhibition of TNFa, IL-1h, IL-6, IL-12, myeloperoxidase and chemokines like CXCL1/Groa, CXCL2/MIP2a, CCL2/MCP1 and CCL3/MIP1a, but VIP increases inflammatory mediators like IL-10 in colon extracts. VIP also reduces the levels of TNFa, IL-1h, IL-6 and serum amyloid A of the treated mice. VIP also modulates the T-cell response, inducing the polarization from a Th1 response to a Th2 response by decreasing the production of IFNg and increasing the production of IL-4 in lamina propria and spleen cell cultures. These results suggest that VIP could be a candidate for the treatment of CD in humans.

VIP inhibits inflammatory mediators in rheumatoid arthritis C. Abad, M.G. Juarranz, C. Martinez, M. Torroba, A. Arranz, J. Leceta, J.L. Pablos, B. Santiago, M. Delgado and Rosa P. Gomariz Faculty of Biology, Department of Cell Biology, Complutense University, Madrid, Spain Rheumatoid artritis (RA) is an inflammatory and autoimmune disease of unknown etiology that leads to chronic inflammation in the joints and subsequent destruction of the cartilage and bone. VIP is an immunopeptide present in the lymphoid microenvironment that elecits a broad spectrum of biological functions, including the modulation of innate and adaptative immunity, showing a predominant anti-inflammatory action. Recently, we have described that VIP significantly reduces the incidence and severity in the mice collagen-induced arthritis model. The therapeutic effects of VIP were associated with the downregulation of both the inflammatory and the autoimmune components of the disease. This study examines the role of VIP in the production of IL-6, TNFa, CXCL8/IL-8 and CCL2/MCP1 in fibroblasts-like synoviocytes cultures obtained from homogenized synovium of arthritic patients. Our results show that VIP inhibits the production of these inflammatory mediators.

The thyroid axis in PACAP-knockout mice is disrupted and contributes to early death Bruce A. Adams, Sarah L. Gray and Nancy M. Sherwood Department of Biology, University of Victoria, Victoria, BC, Canada We have previously reported that mice homozygous for a targeted disruption of the PACAP gene have problems with lipid metabolism and growth culminating in death early in the second post-natal week and this

severe phenotype is temperature dependent. Thyroid hormones (THs) play important roles in development, thermogenesis and growth in mammals. Therefore, we have investigated the thyroidal status of PACAP-knockout mice. Serum levels of total thyroxine (TT4) and total 3,5,3V-triiodothyronine (TT3) were measured in seven-day-old mice. Compared to wildtype mice, serum levels of TT4 were reduced by 25% and TT3 was nearly doubled, suggesting increased conversion of systemic T4 to T3. We found that expression and activity of deiodinase enzymes (responsible for the conversion of THs to active and inactive forms) in brain, liver and brown fat at 7 days are different compared with 2 weeks old or adults. Disruption of the thyroid axis at these early stages in development is critical because the system for managing levels of THs is not mature. Surprisingly, treatment of mice with methimazole (to reduce serum levels of T4 available for conversion to T3) for the first 10 days of life resulted in increased survival of PACAP-knockout mice from 15% to greater than 70%. These data suggest the high levels of serum T3 are contributing to the severe death phenotype. Disruption of the thyroid axis in the PACAP-knockout mouse will be discussed in the context of thermogenesis, stress and growth.

PACAP regulates neurogenic gene expression and modulates the neural lineage in Xenopus laevis embryos D. Alexandre, Z. Hu, V. Lelie`vre, B. Armstrong and J. Waschek Mental Retardation Research Center, Neuropsychiatric Institute, UCLA, Los Angeles, CA, USA The genes encoding PACAP and its selective type I receptor have been characterized in Xenopus laevis, and are expressed in the adult frog brain, but also in embryos at an early stage of development (Hu et al., 2000, 2001). The discrete gene expression patterns of PACAP and PAC1 in the Xenopus embryonic nervous system similar to that in mice and suggest that they might also be involved in Xenopus nervous system development. By microinjecting of mRNA into Xenopus embryos at the four cell stage, we have shown that overexpression of PACAP modulates the expression of genes implicated in the formation of neuronal cells, as confirmed by the decrease of the N-tubulin labelled cells, a specific marker of neurons. To elucidate the molecular mechanism by which PACAP regulate neurogenesis, we have used whole mount in situ hybridization as well as real time PCR to identify N-tubulin upstream target genes of PACAP action. The results indicate that the PACAP regulates the differentiation of neural progenitor cells and could regulate the bifurcation of glial and neuronal fates.

Molecular characterization of a novel variant of type I PACAP receptor which is not coupled to adenylyl cyclase Y. Anouar1, J. Chu1,2, D. Alexandre1, B.J. Gonzalez1, B.K.C. Chow2 and H. Vaudry1 1 Eur. Inst. Pept. Res. (IFRMP 23), Lab. Cell. Mol. Neuroendocrinol., INSERM U413, Univ. Rouen, Mont-St-Aignan, France 2 Department of Zoology, University of Hong Kong, Hong Kong, China

PACAP acts through a type I receptor, PAC1-R, specific for PACAP, and two type II receptors, VPAC1-R and VPAC2-R, that bind PACAP and VIP with high affinity. Several PAC1-R splice variants have been described that presumably fine-tune the effects of PACAP in certain tissues. We have recently identified a novel PAC1-R splice variant in the frog Rana ridibunda. This receptor isoform, named PAC1-Rmc, displays a C-terminal tail whose sequence is distinct from that of the PAC1-R C-terminus due to

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Abstracts

the insertion of a cassette of 13 nucleotides downstream of the sequence encoding the seventh transmembrane domain. PAC1-Rmc is highly expressed in the central nervous system of the frog. However, PACAP failed to activate adenylyl cyclase in cells transfected with PAC1-Rmc. Fusion of PAC1-Rmc with GFP revealed that this receptor, as PAC1-R, is targeted to the plasma membrane. We have recently generated two stable CHO cell lines that express either PAC1-R or PAC1-Rmc. We confirmed in these cell lines the effect of PACAP on adenylyl cyclase through PAC-1R and the lack of effect of the peptide in cells expressing PAC1-Rmc. In addition, we showed that the two receptor variants are able to bind iodinated PACAP with high affinity. Experiments are in progress to determine whether PACAP is able to activate other signaling mechanisms via PAC1-Rmc and whether this variant may act as a negative dominant of PAC1-R, at least in regard to activation of adenylyl cyclase. Supported by INSERM (U413) and a France – Hong Kong exchange program (Procore).

Perspectives on the Development of a Neuroprotective Drug Based on PACAP Akira Arimura US – Japan Biomedical Research Laboratories, Department of Medicine, and Neuroscience Program, Tulane University Health Sciences Center, Belle Chasse, LA, USA PACAP is a pleiotropic neuropeptide, but its neurotrophic and neuroprotective activities may be among its most important activities. Although a number of reports describing PACAP-induced neuroprotection in vitro have appeared, there are considerable differences between these reports in the effective dose as well as in the signaling pathway involved in the neuroprotective action. However, for developing a new drug, it is desirable to conduct such studies under conditions, which are as close to the in vivo conditions as feasible. Then the drug must be shown to be effective in an appropriate animal model, which mimics a human disease. PACAP shows a bimodal neuroprotective dose – response curve in neuron/glia co-cultures, with peaks at 10 13 and 10 10 M. Neuroprotection at 10 13 M involves ERK1/ERK2 activation, while that at 10 10 M involves adenylate cyclase activation. Although PACAP38 has a high efficacy for transport from the blood to the brain across the blood – brain barrier (BBB), it is still difficult to expect that the PACAP concentration in the bran parenchyma will reach greater than femtomolar levels after systemic administration. In another words, cAMP-mediated neuroprotection may not take place in the brain when PACAP38 is given systemically, except in the region where the BBB has been damaged. PACAP38 reduced the infarct size greater than 50% after transient middle cerebral artery occlusion (MCAO) in a rat model of stroke when the body temperature was maintained in the normal range, even when the administration was delayed for 4 h. Although this is a promising finding, it is difficult to expect that PACAP given similarly to stroke patients could also be effective in reducing the infarct by 50% or reducing other symptoms, because of the considerable variation in the magnitude of the insult as well as the normal variations between individual patients. An effort must be made to produce a much greater neuroprotective effect first in a standard animal model, such as an 80% or greater reduction of the infarct size. Studies with PACAP suggest the involvement of various factors in neuroprotection, including stimulation of the production of a neuroprotective growth factor by astrocytes and suppression of iNos activity in microglia, which are induced at femtomolar concentrations of PACAP. Although other mechanisms of action, such as suppression of the inflammatory reaction, including suppression of TNFa expression, have been document for PACAP, these effects appear to be induced by nanomolar concentrations of the peptide. Therefore, such effects cannot be expected with systemic administration. Brain damage induced by stroke involves numerous biological events at the cellular and molecular levels. It is difficult to expect that PACAP alone can counteract all of these reactions. Drug development may have to be directed toward the combined use of two or more neuroprotective agents with different mechanisms of action. In terms

of reduction of the infarct size, we should aim at achieving an 80% reduction of the infarct in the animal model of stroke, before starting the clinical study. This study was supported by NIH grant RO1-NS40373.

PTS-6 as an example of the blood –brain barrier regulatory interface William A. Banks GRECC-St. Louis and Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO, USA The blood – brain barrier (BBB) has long been known as an important regulator of the CNS microenvironment. Recent work has shown that its ability to selectively transport peptides and regulatory proteins endows it with an ability to influence communication between peripheral tissues and the CNS. The transporters also offer an opportunity to deliver therapeutics to the brain. PACAP and BBB interactions provide excellent examples of these principles. PACAP38 is transported both into and out of the CNS by a saturable transporter termed peptide transport system (PTS)-6, whereas PACAP27 is only transported in the brain to blood direction. PTS-6 can deliver enough peripherally administered PACAP to the brain to reverse loss of CA1 hippocampal neurons induced by four vessel occlusion. PTS-6 responds to spinal cord injury in a unique pattern unrelated to BBB disruption. Specifically, a decrease in PTS-6 activity attenuated by time distance from brain occurs soon after injury to the lumbar spine. Seven days after injury, an increase in PTS-6 activity occurs throughout the CNS. Cardiac arrest and middle cerebral artery occlusion each have their own unique effects on PTS-6 unrelated to BBB disruption. These findings suggest that the neuroprotective effects of PACAP may be modulated by regulation of PTS-6.

The passage of VIP/PACAP/secretin family across the blood brain barrier and binding studies in the brain W.A. Banks1 and D. Dogrukol-Ak2 1 GRECC, Veterans Affairs Medical Center and St. Louis University, School of Medicine, Division of Geriatrics, Department of Internal Medicine, St. Louis, MO, USA 2 Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskisehir, Turkey

In recent years, the VIP/PACAP/secretin family has a special interest. The family members that are released into blood from peripheral tissues, especially gut, circulate to the brain and can cross the blood brain barrier. Otherwise, some of the members of this family that originate in the brain and can cross from brain to blood to reach peripheral targets by combination of nonsaturable and saturable transport mechanisms. The binding studies can also clarify the possible site of actions including the identification and isolation of receptors and transporters. The passage of VIP/ PACAP/secretin family could expand our horizons for understanding the CNS effects of peripherally administrated peptides.

Localization and characterization of PACAP receptors in the human cerebellum during development M. Basille1, D. Vaudry1, A. Schilliger1, A. Falluel-Morel1, D. Beaujean1, P. Freger2, N. Gallo-Payet3, H. Vaudry1 and B.J. Gonzalez1 1

IFRMP23, INSERM U413, Rouen University, Rouen, France Department of Neurosurgery, Charles-Nicolle Hospital, Rouen, France 3 Department of Endocrinology, Sherbrook University, Quebec, Canada 2

There is now clear evidence that, in rats, PACAP, acting through PAC1 receptors, plays a pivotal role in the ontogenesis of the cerebellar cortex. In contrast, little is known regarding PACAP receptors in the developing

Abstracts human cerebellum. We have investigated the distribution of PACAP receptors by quantitative autoradiography and RT-PCR in the human cerebellum from 16WEPC to adulthood. At 16WEPC and 21WEPC, a high density of 125I-PACAP27 binding sites was observed in the germinative neuroepithelium, the external granule cell layer (EGL). At 24WEPC, a high concentration of PACAP binding sites was still detected in the EGL but also in the internal granule cell layer, while moderate labeling was found in the molecular layer. At 7Y, 12Y and 22Y, when cerebellar neurogenesis is completed, the density of PACAP receptors was markedly reduced in all cerebellar layers. Competition studies revealed that PACAP38 and PACAP27 were equally potent in displacing 125I-PACAP27 from its binding sites, while VIP was a poor competitor. No significant changes in IC50 values were noticed in the various layers throughout development. Two variants of PAC1 were detected by RT-PCR from 16WEPC to adulthood. The ratio between these two variants changed with development. VPAC1-R was expressed at all stages while VPAC2-R was not detected. These data support the concept that PACAP may exert a neurotrophic effect in the human cerebellum during development, as previously described in rodents. Supported by INSERM.

Delivery of therapeutics to the CNS: the problems and the possibilities David J. Begley Centre for Neuroscience Research, Guy’s Campus, Kings College London, SE1 1UL, UK The blood – brain barrier (BBB) forms a formidable obstacle to the delivery of therapeutics to the central nervous system (CNS). Thus, although neurological diseases as a whole, both acute and chronic, are a major cause of human suffering (WHO Statistics) the treatment of many of these diseases is relatively poorly met because of the difficulties of targeting the required drugs to the CNS. For polar drugs, free diffusional access is effectively prevented by the presence of tight junctions between the cerebral endothelial cells. Molecules which are lipophilic may passively permeate through the BBB-dependant upon their physicochemical properties. Some lipid soluble drugs and their metabolites are substrates for multi-specific efflux pumps (the ABC cassette of transporters) and are actively transported out of the CNS. Some drugs have however been developed, in a largely serendipitous way, that are able to use endogenous transporters present in the BBB in order to enter the CNS. The function of the BBB will be reviewed together with some strategies for optimising CNS drug delivery. At present, a number of strategies for delivering therapeutics to the brain exist or are under development. These include: informed drug design and the optimisation of physico-chemical properties, inhibition of various CNS efflux mechanisms, the development of prodrugs as chemical delivery systems, direct intracerebral injection or infusion, access via the olfactory route, direct modulation or opening of the BBB, delivery via endogenous transporters and also the use of various vector systems including cell penetrating peptides nanoparticles and liposomes.

VIP (vasoactive intestinal peptide) as a modulator of immune and endocrine responses during LPS-induced acute inflammation W. Bik1,2, E. Wolinska-Witort2, M. Chmielowska2, A. Bik1, E. RusieckaKuczalek1 and B. Baranowska2 1 Dept. of Int. Med. Haemataology and Endocrinology, Central Clin. Hosp. Min. of Home Affairs and Adm., Warsaw, Poland 2 Neuroendocrin. Dept. Medical Centre of Postgrad. Education, Warsaw, Poland

VIP may play an important role in endocrine and immune systems.

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The aim of the study was to estimate the influence of VIP’s administration on serum cytokines and pituitary, adrenal, gonadal and thyroid hormones concentrations during LPS-induced acute inflammation. Methods: Male rats Wistar – Kyoto were divided into four groups and each group received respectively placebo (0.9% NaCl), LPS, VIP, LPS + VIP. TNFa, IL6, IL10 and hormones were measured after 2 and 4 h. Data were analysed by Mann – Whitney test. Results: In group which received LPS + VIP compared to the group received only LPS, we found the reductions of: serum IL6 concentration ( p < 0.001) after 2 h and serum corticosterone concentration after 2 and 4 h ( p < 0.05, p < 0.05) as well as the fall in T3, T4 ( p < 0.05, p < 0.05) and testosterone ( p = 0.05) serum concentration after 4 h. We conclude that VIP modulates the immune response through decreasing in proinflammatory cytokines concentration as well as endocrine response in the time of acute inflammation.

Inhibitors of cysteine and aspartic proteases differentially block the survival-promoting activity of various components of a neurotrophic factor complex regulated by VIP D.E. Brenneman1, C.Y. Spong1,2, J. Hauser1, R. Castellon1, J.M. Hill1 and I. Gozes3 1

Lab. Dev. Neurobiol., NICHD, NIH, Bethesda, MD, USA Pregnancy and Perinataology Branch, NICHD, NIH, Bethesda, MD, USA 3 Dept. Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 2

VIP exhibits neuroprotective actions through the release of glia-derived substances including activity dependent neurotrophic factor (ADNF), a 14kDa protein complex. The ADNF complex was separated into three components by N-CHO capillary electrophoresis. The components had similar efficacy but varied significantly (8 orders of magnitude) in their relative potency in preventing neuronal death produced by tetrodotoxin in cell culture. Based on low sequence homology of ADNF digest peptides with known proteases, the mechanistic nature of the components of the ADNF complex was investigated with inhibitors of aspartic (pepstatin A) and cysteine proteases (E64). The most potent (component I) and the least potent (component III) constituents of the ADNF complex were blocked by E64. In contrast, component II was blocked by pre-incubation with pepstatin A, but not E64. Furthermore, proteolytic activity of component II was calcium-dependent and linear with time as shown in a cell-free assay. However, the protease activity exhibited a bell-shaped dose response curve as a function of enzyme concentration. This protease response curve coincided closely with the dose response curve of the survival-promoting activity for component II. These data indicate that at least two proteases are obligatory to the survival-promoting action of the ADNF complex released by VIP. Supported by NICHD, BSF, Neufeld Award, the Gildor Chair.

The effect of PACAP on rhythmic melatonin release of avian pineals V. Csernus1, R. Jo´zsa1, D. Regl} odi1, T. Hollo´sy1, A. Somogyva´ri-Vigh2 and A. Arimura2 1 2

Dept. of Anatomy, Pe´cs Univ. Med. Fac., Hungary US – Japan Biomed. Res. Labs., Tulane University, New Orleans, LA, USA

PACAP is known to participate in the regulation of the circadian rhythm. In the present study, its role in the control of rhythmic melatonin (MT) secretion from chicken pineal gland (ChPG) was investigated. RIA analysis revealed 40 ng PACAP/g ChPG tissue with no midday – midnight differences. Immunohistochemical studies showed PACAP immunoreactive nerve fibers in the perivascular space and around the pinealocytes. Neither

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Abstracts

PACAP-labeled perikarya nor PACAP immunopositivity inside the pinealocytes could be detected. In 5-day perifusion experiments, carried out under darkness, both MT and cAMP showed circadian rhythmic release pattern from explanted ChPG. One hour exposure of ChPG to PACAPinduced transient (3 – 4 h) elevation of MT and cAMP release. The responses were dose dependent in the range of 1 – 100 nM PACAP concentrations. The magnitude of the response was independent of the time of the day in which PACAP was applied. Both normal cAMP cycle and PACAP-induced cAMP responses always preceded similar changes of MT by about an hour suggesting that cAMP is an intracellular intermediate in controlling MT release from the ChPG. At the same time, PACAP did not shift the phase of the in vitro circadian MT rhythm of the pineals. Our data reveal that PACAP is present in nerve fibers in the chicken pineals and pineal cells contain functioning PACAP-sensitive receptors. PACAP apparently exerts a modulatory effect of the rhythmic MT release from the chicken pineal but does not modify the intrinsic biological clock in the avian pineal gland. Supported by OTKA 029553, 032523, 034491 and MTA.

Effects of peptide histidine –isoleucine (PHI), peptide histidine – methionine (PHM) and helodermin on cAMP production in rat and chicken brain

culture model, Xenopus spinal neurons, which highly express PAC1 (>90%). Over 30 min of PACAP gradient exposure, >70% of growth cones grew and turned toward the peptide source, with a turning angle of 20j. Responses were mediated via PAC1, since VIP was ineffective, whereas selective agonist, maxadilan, reproduced effects. Growth cone turning depended on localized cAMP signaling since turning was blocked by cAMP antagonist, PKA inhibitor and increasing cAMP globally with forskolin, whereas attraction was reproduced by focal photolytic release of caged cAMP in growth cones. On the other hand, blockade of Ca2 +, PLC or PI3K signaling pathways did not affect growth cone turning, suggesting the PAC1 short receptor isoform mediated effects. Finally, PACAP-induced attraction involved Rho family of small GTPases and required protein synthesis, recently identified regulators of cytoskeletal directional motility in response to several extracellular signals. In aggregate, these results establish cAMP signaling as an independent pathway capable of mediating growth cone attraction, and raise the possibility that gradients of PACAP from postmitotic layers elicit directional growth from new neurons generated in the proliferative ventricluar zones of the developing embryo.

NAP, a peptide derivative of the VIP-regulated gene ADNP, confers neuroprotection through microtubule dynamics

Agnieszka Djeda1, Izabela Matczak1 and Jerzy Z. Nowak1,2 1

Dept. of Biogenic Amines, Polish Acad. Sci., Lodz, Poland 2 Dept. of Pharmacology, Med. Univ., Lodz, Poland Secretin/glucagon/VIP/PACAP family embraces structurally related peptides, such as PHI, PHM and helodermin (Hd). These peptides display some amino acid similarities to VIP and may act through common receptors. Like VIP and PACAP, PHI/PHM is widely distributed in the body tissues. Still, its role remains largely unknown. Our recent studies concerning biochemical effects of VIP and PACAP in the CNS of rat and fowls have shown their potent stimulatory action on cAMP generation. As PHI/PHM displays some affinity to [125I]VIP-labeled receptors and Hd to VPAC1 type receptors, we asked whether PHI (rat and porcine), PHM and Hd affect cAMP production in [3H]adenine-prelabeled slices of rat and chick cerebral cortex (CCx) and hypothalamus (HTh). All tested peptides (0.3 – 5 AM) dose-dependently stimulated cAMP production in rat tissues, displaying the following rank order of potency: CCx-HdHpPHI > PHM c rPHI; HTh-pPHI>Hd>rPHI>PHM. In chick tissues, PHI and PHM showed none or only weak activity, while Hd dosedependently stimulated cAMP synthesis both in HTh and CCx. A D-Phe4 derivative of pPHI up to 5 AM was without any significant effect on the nucleotide formation in any analyzed tissue, while PACAP38 used at 0.1 AM was a strong stimulator of cAMP synthesis in all experiments. Our results show that receptors mediating some biological effects of PHI/PHM in the CNS can be coupled to AC/cAMP in rats, but not in chicks. Since PHI recognizes specific VIP/PACAP receptors in chick CCx, further study is needed to specify the signaling system through which this peptide acts in the avian brain.

Extracellular gradients of PACAP elicit attractive guidance of neuronal growth cones through localized increases in cAMP signaling E. DiCicco-Bloom, C. Guirland, K.B. Buck, J.A. Gibney and J.Q. Zheng Neuroscience and Cell Biology, Robert Wood Johnson Medical School, NJ, USA

I. Divinski, A.D. Spier and I. Gozes Clin. Biochem., Sackler Medical School, Tel Aviv University, Tel Aviv, Israel Activity-dependent neuroprotective protein (ADNP), a VIP-responsivegene, contains a short bioactive peptide NAP (JPET 293: 1091, 2000). NAP was recently shown to potently protect neurons against a wide variety of insults in vivo and in vitro and may mediate, in part, VIP neuroprotection. Here, survival assays indicated cellular specificity as NAP (10 17 – 10 10 M) protected against oxidative stress in a selected cell line of a neuronal lineage. Affinity chromatography of brain extracts identified tubulin, the brain major protein, as the NAP-binding ligand. Dot blot analysis with pure tubulin and biotinylated NAP verified this binding. When added to cerebral cortical astrocytes, NAP (10 15 – 10 10 M) induced a rapid microtubule re-organization into distinct microtubular structures that were stained by monoclonal tubulin antibodies (PNAS 79: 2579, 1982) and visualized by confocal microscopy. Fluoresceine-labeled NAP induced a similar change and was detected in the intra-cellular milieu, even when cells were incubated at 4 jC or at low pH. The same microtubule reorganization characteristics were observed in cerebral cortical neurons as well as in cell lines of neuronal lineage, but not in cells that were not protected by NAP. Thus, NAP may cross the plasma membrane and interact directly with tubulin, the microtubule subunit, to induce reorganization and survival. As microtubules are a key component of the neuronal and glial cytoskeleton that regulate cell division, differentiation and protection, this finding may explain the breadth and efficiency of the neuroprotective capacities of NAP. Support: ISOA, Gildor Chair, BSF, Neufeld award, ISF, Allon Therapeutics.

Exploration of the ligand binding domains of the secretin receptor using amino-terminal photolabile analogues M. Dong, D.I. Pinon and L.J. Miller Mayo Clinic, Scottsdale, AZ 85259, USA

Developing axons are guided to appropriate targets by environmental cues activating surface receptors and intracellular pathways. Since PACAP receptor, PAC1, is expressed almost ubiquitously in developing nervous system and PACAP is most prominent in postmitotic regions of differentiation, we examined effects on outgrowth and guidance in a well-defined

Photoaffinity labeling is a powerful tool for exploring the molecular basis of interaction between a ligand and its receptor. Using this approach, we have previously demonstrated that probes having a photolabile residue in positions 6, 13, 18, 22 and 26 of secretin all labeled the distal end of the

Abstracts amino terminus of this receptor. While this supports a critical role of this receptor domain in ligand binding, it does not explain the mechanism of activation of the receptor. In this work, we attempted to develop probes having their photolabile residues at the amino terminus of secretin and to use them to explore their approximations with this receptor. Since secretin residue His1 is critical for function, we sited a photolabile benzoylphenylalanine as an amino-terminal extension of the peptide, in position1. We synthesized an additional probe by acetylation of the amino group of this peptide. Both analogues were shown to be full agonists, bound specifically and saturably and were able to efficiently label the secretin receptor. Sequential purification and chemical and enzymatic cleavages of the labeled wild type and mutated secretin receptor demonstrated that the site of covalent attachment with the amino terminally unblocked probe was shown to be in a region that is clearly distinct from that previously labeled. This represented a region spanning the top of the sixth transmembrane domain and the beginning of the third extracellular loop of the secretin receptor. However, the amino-terminally blocked probe labeled a specific residue within the distal amino-terminal tail of its receptor. These data provide important new insight into a possible molecular mechanism for receptor activation.

Light-induced phase shift in the Syrian hamster is attenuated by the PACAP receptor antagonist PACAP6 – 38 or PACAP immuno-neutralization J. Fahrenkrug, A.L. Bergstro¨m, J. Hannibal and P. Hindersson Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted by light via the retinohypothalamic tract (RHT). The RHT contains two neuro-transmitters, glutamate and PACAP which mediate the phase shifting effects of light on the clock. We have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and showed that (1) light-induced phase delay of running wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6 – 38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebro-ventricularly before light stimulation. (2) PACAP administered close to the SCN was able to phase delay the circadian rhythm of running wheel activity similar to light. (3) PACAP is present in the hamster RHT colocalised with melanopsin, a recently identified circadian photopigment. The findings indicate that PACAP is neurotransmitter in the RHT mediating photic information to the clock via the photopigment melanopsin which is located exclusively to the plasma membrane of PACAP expressing cells.

Opposite regulation of the apoptotic mitochondrial pathway by PACAP and C2-ceramide through a map-kinase-dependent mechanism A. Falluel-Morel,1 N. Aubert,1 D. Vaudry,1 M. Basille,1 A. Fournier,2 H. Vaudry1 and B.J. Gonzalez1

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mitochondrial integrity and blocked cytochrome-c release. Q-RT-PCR experiments and Western blot analysis revealed that C2-ceramide induced a time-dependent expression of Bax. PACAP significantly reduced the action of C2-ceramide on Bax and induced Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked respectively by JNK and MEK inhibitors. In addition, the JNK inhibitor blocked the stimulatory effect of C2-ceramide on caspase-9 and -3. A caspase-9 inhibitor as well as the JNK inhibitor prevented C2-ceramide-induced granule cell death. In contrast, the MEK inhibitor U0126 reduced PACAP-induced Bcl-2 expression and promoted apoptosis. Altogether, these data suggest that C2ceramide and PACAP exert opposite regulation of Bax and Bcl-2 expression through JNK- and ERK-dependent mechanisms. Regulation of the mitochondrial apoptotic pathway by endogenous ceramide and PACAP may contribute to the fine-tuning of PCD during neurodevelopment. Supported by INSERM and LARC Network.

Effects of PACAP in a rat model of diffuse axonal injury ´ . Pe´terfalvi1, D. Regl} O. Farkas1, A. Tama´s2, A. Zsombok1,3, A odi2, A. Bu¨ki1, 2 1 4 I. Lengva´ri , T. Do´czi and J.T. Povlishock 1

Dept. of Dept. of 3 Dept. of Hungary 4 Dept. of VA, USA 2

Neurosurgery, Pe´cs Univ. Med. Fac., Hungary Anatomy, Pe´cs Univ. Med. Fac., Hungary Central Laboratory of Animal Research, Pe´cs Univ. Med. Fac., Anatomy and Neurobiology, Virginia Commonwealth University,

Since its discovery, several studies have shown the neuroprotective potential of PACAP. It reduces ischemic death in rat models of both global and focal ischemia. After experimental traumatic brain injury, mRNA of endogenous PACAP is elevated. The aim of the present study was to examine the neuroprotective potential of PACAP in a rat model of diffuse axonal injury. In the first study, rats were treated with a single iv bolus of 125 Ag/kg PACAP or vehicle 5 min before injury. In the second study 1, 10 or 100 Ag PACAP or vehicle was administrated icv after injury, either in a single injection 5 min post-injury, or in two equal amounts 5 and 30 min post-injury. Immunohistochemistry with anti-h amiloid precursor protein (APP), a marker of altered axoplasmatic transport was used to assess axonal damage in the corticospinal tract (CSpT) and the medial longitudinal fascicle (FLM). Results of the first study showed no significant differences 2 or 6 h post-injury in the density of APP-reactive axon profiles in the PACAP- or vehicle-treated rats. In the second study, the density of damaged axons in the CSpT of animals treated icv with 100 Ag PACAP post-injury was significantly lower than that of controls. There was no difference between groups treated with one or two injections. Our results indicate that PACAP might have a protective potential in diffuse axonal injury, but further studies are required to find the optimal administration of PACAP. Supported by OTKA 035195, 034491, ETT, Bolyai Scholarship, MTA, Fogarty Award and NKFP.

Immunohistochemical observation of PACAP and its receptor in the rat testis H. Funahashi1, S. Kato1, S.Nakajo2, R. Suzuki1, A. Arimura3 and S. Shioda1,3

1

IFRMP 23, INSERM U413, Rouen University, Rouen, France 2 INRS-Institut A. Frappier, University of Que´bec, Montre´al, Canada

1

Dept. of Anatomy, Showa Univ. Sch. Med., Tokyo 142-8555, Japan Lab. of Biol. Chem., Showa Univ. Pharm. Sci., Tokyo 142-8555, Japan 3 US – Japan Biomed. Res. Lab., Tulane Univ. Hebert Center, Belle Chasse, LA 70037, USA 2

Ceramides are second messengers involved in programmed cell death (PCD) during development. In the immature cerebellum, ceramides promote granule cell death through an apoptotic process involving caspase-3. PACAP and PAC1-R are actively expressed in immature cerebellar granule cells and PACAP has been shown to prevent PCD by inhibiting caspase-3. We observed that C2-ceramide induced a rapid and marked inhibition of mitochondrial membrane potential and provoked the release of cytochromec in the cytosol. Co-incubation of granule cells with PACAP restored

The rat testis is shown to reveal high level of PACAP immunoreactivities, and the total amount of PACAP in both testes exceed the entire brain in rat. Autoradiography has demonstrated that specific, high-affinity PACAPbinding cites are localized in some of the seminiferous tubules. In this study, to gain insight into the role of PACAP in the rat testicular function, we

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examined the localization of PACAP receptor (PAC1-R) with immunocytochemistry. Then we used a double immunofluorescence method with antiPAC1R and anti-PACAP antibodies to examine precisely the stages of PACAP immunoreactivities in the testicular germ cells. PAC1-R immunoreactivity was detected in the developing germ cells but not in either Sertoli or Leydig cells. Strong PAC1-R immunoreactivity was detected in the spermatids from cap stage to acrosome stage but not in the maturation stage. Double-immunostaining method revealed that both immunoreactivities of PACAP and PAC1-R were detected in the acrosome in developing spermatids. These findings strongly support the notion that PACAP participates in spermatogenesis, particularly in spermiogenesis. Moreover we are now trying to identify ultrastructural localization of PACAP and PAC1-R in order to find out PACAP action cites in the spermatids.

Characterization of the testis-specific promoter region in human pituitary adenylate cyclase-activating polypeptide (PACAP) gene T. Futagawa1, H. Sugahara1,2, M. Ushiyama1,2, K. Yamada1 and A. Miyata2 1 Dept. of Clinical Pharmacy, Kagoshima Univ. Fac. Med., Kagoshima 8908520, Japan 2 Dept. of Pharmacol., Kagoshima Univ. Fac. Med., Kagoshima 890-8520, Japan

PACAP is a member of the glucagons-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high levels in the germ cells of the testis, where it locally activates cAMP-coupled receptors located in the somatic Sertoli cells. Recently, it has been reported that a single PACAP gene uses a testisspecific promoter to express the mRNA containing a unique exon in rat (Daniel et al., 2000). By searching the human genome sequence-database, we have identified a human homologue of testis-specific exon of PACAP gene. The potential testis-specific exon is localized 10.9 kb upstream from the first exon. In RT-PCR of human testicular RNA, we have confirmed the expression of testis-specific variant of the PACAP mRNA. The human testis-specific promoter region contains a number of possible binding sites for transcriptional regulation factors (AML-1a, SRY, OCT-1, AP-1, GATA1, etc.). Further the analysis of the promoter activity is in progress now. The testis-specific regulatory mechanism of PACAP gene expression will be also discussed.

PAC1 and PACAP stimulate signaling and growth in human colonic cancer cells Patrizia Germano, Sang Le and Joseph Pisegna

ry: These data indicate that HCT8 human colonic tumor cells express biologically active PAC1 receptors and PACAP, suggesting a potential autocrine mechanism. PAC1 internalize within 15 min from PACAP-38 addition. Stimulation with PACAP-38 activates intracellular signaling through adenylyl cyclase and causes an increase in [Ca2 +]i. In addition, a 25% increase in growth was observed in PACAP-38 stimulated HCT8 cells, whereas no increase was detected with VIP, indicating that the effects were mediated by PAC1.

PAC1 internalize and modulate TCR-induced signal transduction in Jurkat cells P.M. Germano*, D.S. Oh, S. Lieu, K. Tachiki and J.R. Pisegna VA GLAHS, and UCLA School of Medicine, CA 90073, USA Introduction: We previously showed PAC1 expression on primary T cell lines and on Jurkat cells. Hypothesis: PACAP activates predominantly the cAMP pathway, inhibiting the IL-2 synthesis and the PI3-K-Akt oncogenic pathway, with consequent anti-proliferative effects, whereas in CD3-activated cells, triggering of PAC1 and TRC enhances intracellular Ca2 + release, PKC activation and inhibition of apoptosis. Methods: FluorPACAP and the rabbit polyclonal anti-PAC1 were used for internalization studies by confocal microscopy. cAMP release was measured by ELISA. Ca2 + signaling was studied in the presence or absence of anti-CD3. Results: Internalization of Fluor-PACAP occurred at 30 s and was nearly complete within 10 min. Anti-PAC1 Abs confirmed PAC1 internalization. cAMP was stimulated in a dose-dependant fashion with an efficacy fourfold over basal. Fluo-4-loaded cells demonstrated coupling between PAC1 and [Ca2 +]i only in CD3-activated cells. Cytosolic elevation of [Ca2 +]i upon anti-CD3 activation was enhanced by PACAP-38. PACAP-38 was not able to modify the [Ca2 +]i cytosolic levels when added to TCR-inactivated Jurkat cells. VIP did not influence these responses. Conclusions: PAC1 expressed in Jurkat cells are functional and internalize in a manner similar to PAC1 transfected in NIH-3T3 cells. PAC1 couples only to the cAMP pathway, but not to intracellular [Ca2 +]i elevation, whereas it enhances CD3-induced [Ca2 +]i responses. These results suggest that PAC1 acts as a co-receptor during TCR activation in T cells, leading to a downregulation of antigen-induced apoptosis and increase of proliferative responses, whereas it couples only to cAMP in absence of TCR triggering.

PACAP modulates expression of sympathetic neuron transcripts associated with nervous system development and functions B.M. Girard, K.N. Dozark, V. May and K.M. Braas

UCLA, Los Angeles, CA, USA Background: We have previously demonstrated that PAC1 has is expressed on human colon cancer cells lines although little is known about its signaling and proliferative effects. Aims: PACAP and PAC1 expression, internalization, signal transduction and role in regulating growth of HCT8 human colonic tumor cells were investigated. Methods: RT-PCR, immunofluorescence staining and FACS analysis were performed. Fluor-PACAP and anti-PAC1 were used to characterize PAC1 internalization. Induction of cAMP was determined by dowex/alumina chromatography. [Ca2 +]i response to PACAP-38 was determined by Dynamic video imaging. MTT assay was performed to study HCT8 growth indexes. Results: Immunofluorescence and FACS analyses with anti-PAC1 and anti-PACAP Abs showed PAC1 and PACAP expression. RT-PCR further confirmed the expression of PAC1. By confocal imaging, Fluor-PACAP and anti-PAC1 revealed PAC1 internalization between 1 and 15 min. PACAP-38, dose-dependently, elevated cAMP levels with an EC50 of 3.0 nM and an induced an increase in [Ca2 +]i, partially inhibited by the PACAP(6 – 38) antagonist. Furthermore, PACAP-38 stimulated HCT8 growth upon 24-h activation. Summa-

Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA PACAP peptides have well-described neuro-modulatory and neurotrophic activities. Sympathetic postganglionic neurons of the rat superior cervical ganglion (SCG) express PAC1(short)HOP1 receptors which engage adenylyl cyclase, PLC, MEK/ERK and PI3K/Akt signaling pathways. PACAP activation of PAC1R through these mechanisms depolarizes SCG neurons, and stimulates catecholamine and NPY production/secretion. Recently, we have shown that PACAP peptides augment SCG neuromodulatory peptides. Using RIA and real-time quantitative PCR, PACAP augmented time- and concentration-dependent SCG neuron VIP, PACAP, galanin, and substance P peptide and transcripts levels. The PAC1R-selective responses were specific for PACAP, and blocked by the antagonists PACAP(6-38) and MaxD4. Selective inhibitors, including H89, BimI, PD98059 and LY294002, preferentially blocked the modulation of specific neuropeptides, supporting PACAP/PAC1 receptor activation of multiple signaling cascades. Complementary SCG neuron microarray studies corroborated these results, and

Abstracts identified additional neuropeptides increased by PACAP. The microarray analyses showed that expression of other SCG transcripts were also altered by PACAP, including many transcriptional factors and accessory proteins, vesicular components, cytokines/growth factors and receptors, and ion channels, necessary for neuronal survival, differentiation and function. Supported by NIH HD27468, NS37179 and P30CA22435, and AHA 0120367T.

Molecular determinants and immune consequences of the T cell-targeted VPAC2-transgenic (TG) mouse phenotype

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ciency, cholinergic toxicity and excess amyloid precursor protein expression. These results lead from basic VIP research to potential drug design. Support: ISOA, Gildor Chair, BSF, Neufeld award, ISF and Allon Therapeutics Inc.

The effect of selective VPAC ligands on tone and transmural nerve stimulation in porcine cerebral arteries S. Grant1, E.M. Lutz2, P. Robberecht3 and R.M. Wadsworth1 1

Dept. of Physiol. and Pharmacol., Univ. Strathclyde, UK Dept. of Biosci. and Biotech., Univ. Strathclyde, UK 3 School of Medicine, Univ. Bruxelles, Brussels, Belgium 2

Edward J. Goetzl, Glenn Dorsam, Carola Grinninger and Julia K. Voice University of California, San Francisco, CA 94143-0711, USA Constitutive expression of high levels of the usually inducible VPAC2 receptors by thymocytes and CD4 T cells of VPAC2 TG mice results in elevated blood levels of IgE and eosinophils, strikingly increased immediate-type hypersensitivity (ITH) and decreased delayed-type hypersensitivity (DTH). This VPAC2 TG phenotype is driven by a high Th2/Th1 cytokine ratio, which is attributable principally to an elevated number of IL-4secreting CD4 T cells. The high Th2/Th1 cytokine ratio is dependent on CD4 T cell-derived VIP and is mediated by c-maf, but not t-bet-1 or gata-3. Other immunologic characteristics of the VPAC2 TG phenotype are due to increased VPAC2 effects on subsets of CD4 T cells, such as CD4+CD25+ T cells. Although less is known of the mechanisms underlying increased DTH and decreased ITH in VPAC2-null mice, there is an increased Th1/Th2 cytokine ratio. The VPAC2-null mouse immune phenotype is not dependent on endogenous VIP, but instead is partially reversed by additional VIP. Exogenous VIP in the VPAC2-null mouse reduces the Th1/Th2 cytokine ratio, decreases CD4 T cell production of IFN-g and increases the number of IL-4-secreting CD4 T cells by presumed signaling through T cell VPAC1 receptors. Thus, the VIP-VPAC receptor axis is a highly plastic system for regulation of effector CD4 T cell functions.

VIP and PACAP regulate activity-dependent neuroprotective protein (ADNP), a gene that shapes and protects the brain 1

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I. Gozes , A. Pinhasov , S. Mandel , I. Vulih , M. Zusev , R.A. Steingart , E. Giladi1 and D.E. Brenneman2 1

Department of Clinical Biochemistry, Sackler Medical School, Tel Aviv University 69978, Israel 2 SMDP, LDN, NICHD, NIH, Bethesda, MD 20892, USA We have recently cloned the activity-dependent neuroprotective protein (ADNP) gene and revealed that it is regulated by VIP in rat cortical astrocytes. We now showed that: (1) astrocyte ADNP was regulated by PACAP and VIP in a time dependent manner, through the VPAC2 receptor. (2) ADNP knockout embryos exhibited cranial neural tube closure failure and died on E8.5 – 9.0, a time when VIP functions as an embryonic growth factor while augmenting ADNP synthesis. (3) Oct4, a gene associated with germ-line maintenance was markedly abundant, while Pax6, a gene crucial for cerebral cortex formation, was not detected in the brain primordial tissue of the knockout embryos, in marked contrast to normal embryos. (4) Heterozygous ADNP /+ male mice exhibited growth restriction and impaired learning. (5) Recombinant human ADNP protected pheochromocytoma (PC12) cells against h amyloid peptide toxicity and oxidative stress, through the regulation of the p53 gene and genes related to the NFk-B family. (6) Peptide scanning of ADNP resulted in the discovery of a short sequence, NAP (NAPVSIPQ) that provides neuroprotection in femtomolar concentrations, mimicking, in part ADNP’s activity. In vivo, NAP protected against learning impairments in animal models of apolipoprotein E defi-

Vasoactive intestinal polypeptide (VIP) is a major cerebrovasodilator neurotransmitter present in cerebral intramural nerves. However, the receptors involved have not been reported and were therefore investigated. The mRNA encoding the VPAC1, VPAC2 and NPR-C receptors were detected by RT-PCR. Pig basilar artery rings suspended in a myograph relaxed to VIP, the selective VPAC1 agonist, [Lys15Arg16Leu27]VIP(1 – 7)/ GRF(8 – 27), VPAC2 agonist (Ro-25-1553) and the NPR-C agonist (cANP(4 – 23)) in a concentration-dependent manner. A total of 100 AM L-NAME reduced the relaxation caused by VIP, and the VPAC1 and NPR-C agonists (two-way ANOVA, p < 0.05, n = 6 – 8), but had no effect on Ro-251553. Additional evidence of receptor involvement was obtained with specific antagonists. The 1 AM [AcHis 1 DPhe 2 K15 R 16 L27 ] VIP(1 – 7)GRF(8 – 27), 0.1 AM PG-99465 and 10 AM ANP(1 – 11) all attenuated the relaxation caused by VIP, demonstrating the involvement of VPAC1, VPAC2 and NPR-C receptors, respectively (two-way ANOVA, p < 0.05, n = 6). Transmural nerve stimulation (100 (0.2 ms) square-wave pulses at 8 Hz) was delivered by platinum electrodes. The resulting relaxation was attenuated by the VPAC2 antagonist, PG-99465. In conclusion, the mRNA of all three known VIP receptors are present in porcine basilar arteries. Relaxation responses to selective ligands in these arteries support that these receptors are present and are functional. Part of the VIP-mediated cerebral vasodilation is linked to nitric oxide and involves VPAC1 and NPR-C receptors. Neurally mediated vasodilation appears to involve VPAC2 receptors.

VPAC receptor expression in mouse splenic CD4 + CD25 + regulatory T cells C. Grinninger, K. B-Oskoui and E.J. Goetzl Medicine University of California, San Francisco, CA, USA VPAC1 and VPAC2 G protein-coupled receptors are prominent in the immune systems of mammals, where they influence diverse T celldependent response including Th1/Th2 differentiation and functions. Little is known about expression and effects of the VIP/VPAC axes in functionally distinctive subsets of Th cells. CD4+CD25+ regulatory T cells are crucial for some types of immunosuppression and induction of immune tolerance. We hypothesized that the CD4+CD25+ subset may express a different profile of VPAC receptors and thus respond uniquely to VIP compared to the universe of CD4+CD25 Th1/Th2 cells. We quantified mRNA levels for VPAC1 and VPAC2 receptors in T cell subsets of wild-type, mVPAC2-null and T cell-targeted hVPAC2 transgenic C57BL/6 mice. Endogenous mVPAC1 mRNA levels in CD4+CD25+ T cells were 25 times higher for hVPAC2-TG mice than for wild-type and VPCA2-null mice, which were similar. In contrast, CD4+CD25- T cells from the same mice showed 20- to 30-fold higher levels of endogenous mVPAC1R expression in wild-type and VPAC2-null mice than hVPAC2-transgenic mice. Endogenous mVPAC2R was not detectable in any CD4+ T cell subsets of wild-type or mVPAC2-null mice, but was found in hVPAC2-transgenic mice, where the level was 4-fold

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higher in CD4+CD25+ T cells than in CD4+CD25- cells. Thus, the human VPAC2 receptor transgene appears to control expression of endogenous VPAC receptors in a pattern suggesting special contributions of CD4+CD25+ T cells to VPAC2 enhancement of the Th2-mediated phenotype of allergic diseases.

Pleiotropic effects of PACAP in differentiating pheochromocytoma cells: a microarray and suppression subtractive hybridization study L. Grumolato1, H. Ghzili1, A.G. Elkahloun2, D. Alexandre1, L. Yon1, L.E. Eiden3, A. Fournier4, H. Vaudry1 and Y. Anouar1 1

Eur. Inst. Pept. Res., Lab. Cell. Mol. Neuroendocrinol., INSERM U413, Univ. Rouen, Rouen, France 2 NHGRI, NIH, Bethesda, MD, USA 3 NIMH, NIH, Bethesda, MD, USA 4 INRS, Univ. Quebec, Montreal, QC, Canada

specific PAC1 receptor as shown by in situ hybridization. PACAP containing retinal fibres were not demonstrated in the superior colliculus or the dorsal raphe nucleus. The findings indicate that PACAP could be a neurotransmitter involved in circadian timing, masking, regulation of sleep – wake cycles and pupillary reflexes. Some of the ChB labelled projections did not contain PACAP suggesting the existence of nonPACAP/non-melanopsin projection(s) to the same areas a notion which may explain why melanopsin deficient mice have retained ability to entrain to light.

Glucose homeostasis and beta-cell proliferation in PACAP transgenic mice H. Hashimoto1, S. Tomimoto1, N. Shintani1 and A. Baba1,2 1 2

Lab. Mol. Neuropharmacol., Grad. Sch. Pharmaceut. Sci. Lab. Mol. Pharmacol., Grad. Sch. Med., Osaka Univ., Osaka, Japan

The neuropeptide PACAP participates to neuroblast proliferation and differentiation. To gain insight into the role of this neuropeptide in the development of sympathoadrenal (SA) cells, we first defined the phenotype of rat pheochromocytoma PC12 cells after differentiation with PACAP. The neuropeptide modified the electrical properties of these cells and regulated the expression of neuronal as well as neuroendocrine gene markers. In order to obtain an overall view of the mechanisms triggered by PACAP during SA cell differentiation, we compared the transcriptomes of rat adrenomedullary chromaffin cells, PC12 cells and PACAP-differentiated PC12 cells, using the complementary techniques of microarrays and suppression subtractive hybridization. Data analysis revealed that among the genes differentially expressed between non-tumoral and tumoral chromaffin cells, a high number is also regulated by PACAP during PC12 differentiation. A large proportion of PACAP-regulated genes is involved in cell proliferation/differentiation, survival and motility/adhesion. Kinetic studies carried out on some of the isolated genes revealed that PACAP exerts early as well as late effects to achieve the genetic program necessary for cell differentiation. In conclusion, this study has identified a large number of PACAP-target genes in PC12 cells, which may mediate the pleiotropic effects of the neuropeptide in differentiating normal or tumoral SA cells.

PACAP is a potent insulinotropic hormone currently under study as a therapeutic agent for diabetes. Using a reverse pharmacological approach, we are investigating the roles of PACAP in insulin/glucose homeostasis. For this purpose, we have generated transgenic mice overexpressing PACAP in the islet beta cells (Tg/+). Tg/+ mice showed an enhanced glucose-induced insulin release compared to wild-type mice, despite a similar glucose response. The PACAP transgene ameliorated streptozotocin-induced diabetes. Notably, an increase in 5-bromo-2-deoxyuridine (BrdU)-positive beta cells in the streptozotocin-treated transgenic mice was observed. Morphometric analysis revealed that total islet mass tends to increase in 12-month-old transgenic mice, but showed no difference between 12-week-old transgenic and nontransgenic littermates. We also crossed lethal yellow KKAy mice (Ay/+), a genetic model for obesity-diabetes, and Tg/+ mice. Both Ay/+ mice and Tg/+ mice with the Ay allele (Tg/+:Ay/+) showed similar diabetic phenotypes, however, hyperinsulinemia, which is usually seen in obesity-associated diabetes, were suppressed in Tg/+:Ay/+ mice. In Ay/+ mice, the pancreatic wet weight was increased and islet hyperplasia was observed; however, the islet hyperplasia was significantly reduced in Tg/+:Ay/+ mice. The present data suggest a protective role for PACAP against type I diabetes as well as obesity-associated diabetes, and the possible involvement of PACAP in the beta cell neogenesis. This study also suggests a possible regulatory effect on islet hyperplasia resulting from insulin resistance.

Target areas innervated by PACAP immunoreactive retinal ganglion cells

Effects of ghrelin on GH secretion in ruminants

Jens Hannibal and Jan Fahrenkrug

T. Hashizume1, M. Horiuchi1, S. Nonaka1, E. Kasuya2, M. Kojima3, H. Hosoda4 and K. Kangawa4

Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark The retinohypothalamic tract (RHT) originates from a subset of retinal ganglion cells (RCGs). The cells of the RHT co-store the neurotransmitters PACAP and glutamate, which mediate light information to the circadian clock located in the SCN. These ganglion cells are intrinsically photosensitive due to expression of melanopsin, a putative circadian photopigment. PACAP and melanopsin are exclusively co-stored in RCGs constituting the RHT. The study examined if PACAP occurs in retinal pathways to the brain not only responsible for light entrainment, but also those involved in masking and control of melatonin secretion and pupillary reflex. Using intravitreal injection of the anterograde tracer cholera toxin subunit B (ChB) and double immunostaining for PACAP and ChB, PACAP containing nerve fibres constitute the major projections to the SCN and the intergeniculate leaflet of the thalamus (IGL) and innervated also the ventrolateral preoptic nucleus, the anterior and lateral hypothalamic area, the ventral part of the lateral geniculate nucleus and the olivary pretectal nucleus. In addition these areas expressed the PACAP

1

Fac. of Agri., Iwate Univ., Japan Lab. Anim. Neurophys., Nat. Inst. of Agrobio. Sci., Kurume Univ., Japan 3 Inst. of Life Sci., Kurume Univ., Japan 4 Dept. of Biochem., Nat. Cardiov. Ctr. Res. Inst. 2

The effects of ghrelin on GH secretion in ruminants were examined in vitro and in vivo. Human ghrelin (hGhrelin) and rat ghrelin (rGhrelin) stimulated GH release from cultured bovine anterior pituitary (AP) cells at doses from 10 10 to 10 7 M and from 10 9 to 10 7 M, respectively ( P < 0.05). GHRH increased GH concentrations in cultured media at a dose as low as 10 13 M ( P < 0.05). Somatostatin (SS, 10 7 M) blunted GH release induced by hGhrelin (10 8 M) and GHRH (10 8 M). In the presence of SS, additive effects of hGhrelin and GHRH on the release of GH were observed ( P < 0.05). Intra-arterial injections of rGhrelin (3 Ag/kg BW) increased plasma GH concentrations in goats, but the GH secretory response was weaker than that of GHRH (0.25 Ag/kg BW) ( P < 0.05). Intra-hypothalamic injections of rGhrelin (10 nM/200 Al) increased plasma GH concentrations in cattle. These results show that ghrelin stimulates GH

Abstracts

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release in ruminants; however, the GH-releasing effect is weaker than GHRH. The present results also show that ghrelin interacts with GHRH and SS in the release of GH from bovine AP cells.

PACAP and PACAP receptors mRNA expression in ES cell-derived neural stem cells and in neurosphere cultures

Hypoxic damages of the BBB: An in vitro study with the new BBB model

M. Hirose1,3, H. Hashimoto1, N. Shintani1, N. Arakawa1, J. Iga1 and A. Baba1,2

K. Hayashi1, S. Nakao1, S. Nakagawa1, M. A. Deli1,2 and M. Niwa1

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Dept. Pharmacol., Nagasaki Univ. Grad. School of Med., Nagasaki, Japan Biol. Res. Centr. Hungarian Acad. Sci., Szeged, Hungary

We made a new model of the BBB with brain endothelial cells and pericytes, and checked the effects of hypoxia on the model. As hypoxic damages to the BBB induce vasogenic brain edema, we also investigated the effect of edaravone, an antioxidant of superoxide, on hypoxic BBB damages, with use of our in vitro model under the condition of hypoxia/ reoxygenation. Three types of the BBB model, endothelial cells type (E00), endothelial cells with pericytes (non-contact, E0P) and endothelial cells with pericytes (contact, EP0), were made by culturing these cells on the Transwell co-culture system. We evaluated the BBB functions with transendothelial electrical resistance (TEER), and permeabilities of Evans’ bluealbumin (EBA) and sodium fluorescein (Na-F). The hypoxic condition was made with Anerocult hypoxic kit (Merck). Edaravone was applied at doses of 1 pM – 1 mM. The strongest barrier-function was observed in an EP0. Despite of hypoxia induced-disruption of the barrier in E00 and E0P models, a minimum dysfunction was seen in EP0. Edaravone inhibited hypoxia/reoxygenation induced reduction of TEER, dose-dependently. Hyperpermeabilties of EBA and Na-F were reduced by edaravone. We found that pericytes work well for the BBB. The cells may secrete factors to induce endothelial maturation and keep the BBB functional and stable. We also obtained the evidence that antioxidant edaravone protects endothelial cells from hypoxic injury and keeps the BBB functional.

Neonatal and adult mouse social behaviors disrupted by blockage of VIP during embryogenesis J.M. Hill1, L.R. Powell1, S.Y. Flores1, N.M. Tompkins1, L.M. Sheppard1, I. Gozes2, D. Abebe1 and D.E. Brenneman1 1 2

Lab. of Devel. Neurobiol., NICHD NIH, Bethesda, MD, USA Clin. Biochem., Tel Aviv Univ., Tel Aviv, Israel

VIP has a critical role in CNS development during the early postimplantation period of mouse embryogenesis. Blockage of VIP action at this time results in microcephaly, growth restriction and developmental delays in the neonatal mouse. Further links between VIP and neurodevelopmental disorders are suggested by the dysregulation of VIP in the segmental trisomy model of Down syndrome and the finding that higher levels of VIP occur in neonatal blood spots of babies with autism and Down syndrome. In the current study, we show that neonatal mice experiencing a blockage of VIP during embryogenesis (VA) exhibit a significant increase in latency to contact their mother after separation (VA—100.9 F 26 vs. control—40.7 F 7.8 s) and, in the maternal preference test, only 28% preferred their mother over a strange lactating female, compared with 68% of control pups. As adults, treated mice exhibited increased stereotypy and, unlike control and PACAP-antagonist animals, did not modify their behavior in the presence of a strange animal. Treated animals were less aggressive than control mice and also failed to recognize a strange mouse in the social recognition test. Two of the defining characteristics of autism are increased stereotyped-repetitive behaviors and abnormalities in social behavior. The mouse model produced here shares these important characteristics with autism. These data suggest not only that this paradigm has potential as a model for neurodevelopmental disorders but also that disruption of VIP function during embryogenesis may be a common feature in disordered development.

Lab. of Mol. Neuropharmacol., Grad. Sch. Pharmaceut. Sci., Osaka Univ., Osaka, Japan 2 Lab. of Mol. Pharmacol., Grad. Sch. Med., Osaka Univ., Osaka, Japan 3 JSPS Research Fellow, Japan. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor (PAC1) are expressed widely in the embryonic brain, especially neuraxis, where newborn neurons generate. Therefore, PACAP is considered to play a role in developing of central nervous system. To examine the possible role of PACAP in early neuronal development, in this study, we used (1) embryonic stem (ES) cell-derived neural differentiation system and (2) neurosphere cultures which contain CNS stem cells, and investigated the gene expression during the ES cell-derived neural differentiation and effects of PACAP on proliferation and differentiation in neurosphere cultures. Stage-dependent mRNA expression of PACAP and its receptors was observed during neural differentiation from ES cells. In ES cells, PACAP and PAC1 receptor mRNAs were slightly expressed. The two mRNA expressions were increased in neural stem cell-enriched culture and were markedly upregulated following induction of neuronal/glial differentiation. PACAP has been shown to inhibit mitogenesis of rat cultured cortical precursor cells. In CNS stem cells, PACAP decreased the number of cells expressing the neural stem cell-specific marker nestin. By contrast, PACAP significantly increased the cells positive for the neuronal marker MAP2 without increasing the cells expressing the astrocyte marker GFAP. These results suggest that PACAP inhibits mitogenesis of neural precursor cells but instead promotes the terminal stage of neuronal differentiation.

Effects of in ovo treatment with PACAP antagonist on the neurobehavioral development of chicken T. Hollo´sy1, R. Jo´zsa1, D. Regl} odi1, B. Jakab2, J. Ne´meth2 and I. Lengva´ri1 1 2

Department of Anatomy, Pe´cs University Medical Faculty, Hungary Department of Pharmacology, Pe´cs University Medical Faculty, Hungary

Recent studies show that PACAP plays an important role in the development of the nervous system. The aim of the present study was to investigate the effects of the PACAP antagonist PACAP6 – 38 on the neurobehavioral development of chicken. Chicks were chosen for this study for the possibility of treating embryos in ovo, without the influence of maternal factors. Eggs were treated with 20 Ag PACAP6 – 38 injected into the yolk sac or physiological saline in the first and second half of embryonic life. After hatching, general behavior was tested daily for 2 weeks. Open-field activity was tested on postnatal days 3 and 14. Social behavior was studied with runway test on postnatal days 4 and 15. Our findings showed that animals treated with PACAP6 – 38 in the first half of embryonic life were more active after hatching, as shown by the higher number of steps and escape attempts, and the less time spent inactive in the open-field. Also, more animals in the PACAP6 – 38-treated group showed running and preening activity. This difference disappeared at 2 weeks of age. Animals treated in the second half of embryonic life showed no difference in activity. On the other hand, the runway test showed that antiPACAP-treated animals were less social than control ones. PACAP and VIP levels measured by RIA at 1 month of age slightly differed in certain brain areas between the groups, but this difference disappeared at 5 months of age. Our results show that anti-PACAP has a transient effect on the neural development and provide the background for further investigations. Supported by OTKA 034491, 043467, 032523 and MTA.

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Abstracts

Role of PACAP in secretion of catecholamine from adrenal medullary cells M. Inoue and N. Fujishiro* Dept. Cell and System Physiol. Univ. Occupational Environmental Health Sch. Med., Fukuoka, Japan *Dept. Physiol. Fukuoka Univ. Sch. Med., Fukuoka, Japan The role of PACAP in catecholamine secretion from dissociated adrenal chromaffin cells of guinea-pig and rat was investigated using amperometry and the patch clamp technique. Pretreatment of chromaffin cells with PACAP for 2 min induced a dose-dependent enhancement of secretion in response to nicotine and muscarine. The PACAP pretreatment also enhanced the secretion due to Ca2 + mobilized from internal store sites with no change in time course. Application of PACAP did not change the holding current level at 60 mV or nicotine-induced inward currents. Similarly, preexposure to PACAP did not affect peak amplitudes or decay time courses of outward currents due to Ca2 + mobilization in response to high concentrations of muscarine. PACAP-induced enhancement of nicotineand muscarine-induced secretions was suppressed by the simultaneous application of PACAP and the protein kinase inhibitors. Application of forskolin enhanced both muscarine- and nicotine-induced secretions, whereas application of phorbol12,13-dibutyrate augmented the nicotineinduced secretion, but suppressed the muscarine-induced secretion. These results suggest that PACAP enhances secretion by acting on secretory machinery, but not Ca2 + handling mechanisms and the enhancement is mediated by cAMP-dependent protein kinase.

Expression of mRNA for PACAP and its receptors in human pheochromocytomas K. Isobe1, I. Tatsuno2, K. Takekoshi1 and Y. Kawakami1 1 2

Dept. of Clin. Pathol., Univ. of Tsukuba, Tsukuba, Japan Dept. of Clin. Cell Biol., Chiba Univ., Chiba, Japan

PACAP induces the expression of catecholamine (CA)-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding CA-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and TH and PNMT mRNA in pheochromocytomas. mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH ( p = 0.0018) and PNMT ( p = 0.05) mRNA expression, as well as epinephrine ( p = 0.0342) levels in 16 intra-adrenal pheochromocytomas. Our findings support a possible role for PACAP in the regulation of expression of genes encoding CA-synthesizing enzymes in intra-adrenal pheochromocytomas.

Unsymmetrical abnormalities on phase shift in PACAP knockout mice Y. Isojima1, C. Kawaguchi2, K. Tanaka2, N. Shintani2, H. Hashimoto2, A. Baba2,3 and K. Nagai1 1

Div. Protein Metabolism, Inst. Protein Res., Osaka Univ., Osaka, Japan Lab. Mol. Neuropharmacol., Grad. Sch. Pharmaceutical Sci., Osaka Univ., Osaka, Japan 3 Lab. Mol. Pharmacol., Grad. Sch. Med., Osaka Univ., Osaka, Japan 2

In mammals, the pacemaker of the circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN). The circadian clock is

adjusted to daily day – night cycle via the retinohypothalamic tract (RHT), a direct projection from the retina to the SCN. PACAP is one of the neurotransmitters as well as glutamate in RHT, and has been suggested to be involved in the resetting mechanism of the circadian clock. We studied profiles of the pacap knockout (pacap / ) mice to clarify the role of PACAP in the generation and resetting the circadian clock. Pacap / mice reentrained to 6-h delay and advance of light/dark cycles similarly to wild type mice. Light stimulation at late subjective night resulted in significantly attenuated phase advance in pacap / mice compared with wild type mice. In contrast, light stimulation at early subjective night caused modest reduction of phase delay in pacap / mice against wild type mice. These different effects of PACAP deficiency to phase delay and advance suggest the unsymmetrical roles of PACAP in phase resetting of the circadian clock.

Antisense for met-enkephalin crosses the blood – brain barrier L.B. Jaeger1 and W.A. Banks1,2 1 Saint Louis University School of Medicine, Department of Pharmacology and Physiology, St. Louis, MO, USA 2 GRECC, Veterans Affairs Medical Center-St. Louis and Saint Louis University School of Medicine, Division of Geriatrics, Department of Internal Medicine, St. Louis, MO, USA

Alcohol consumption is associated with complex changes in neurochemistry. In mice, levels of Met-Enkephalin (Met-Enk), an endogenous opiate peptide, increase when the animal is initially exposed to ethanol, however, chronic exposure is correlated with decreased Met-Enk levels. Upon withdrawal from alcohol, Met-Enk levels remain low or decrease even further. Morphine, an opiate agonist, has been shown to increase ethanol consumption in rats while naloxone, an opiate antagonist has the opposite effect. Peptides of the VIP/PACAP/secretin family have been shown to modulate the effects of morphine and ethanol possibly through action on Met-Enk. Treatment of mice with vasoactive intestinal polypeptide (VIP) decreases the analgesic response to morphine and blocks the development of morphine tolerance. PACAP prevents ethanol-induced apoptosis when administered to rat neuronal cell cultures. Furthermore, previous studies have shown that VIP nerve terminals synapse on Met-Enk perikarya and dendrites in the bed nucleus of the stria terminalis (BST), a relationship which suggests a modulatory role of VIP on the activity of Met-Enk neurons. To better understand the complex interactions between these neuropeptides, we have developed three antisense phosphorothiolate oligodeoxynucleotides (ODNs) against Met-Enk. The ODNs were labeled with P32 and administered separately to mice via i.v. injection. All three ODNs were transported intact across the blood – brain barrier by a non-saturable system, decrease brain Met-Enk levels and increase ethanol consumption.

Discovery of ghrelin: its structure and functions K. Kangawa Department of Biochemistry, National Cardiovascular Center Research Institute, Japan Small synthetic molecules termed growth hormone secretagogues (GHSs) act through an orphan G-protein coupled receptor, GHS-R, to stimulate growth hormone (GH) release from pituitary. To search for the natural ligand for GHS-R, we constructed a stable CHO cell line expressing rat GHS-R for monitoring intracellular calcium concentration [Ca2 +]i change induced by rat tissue extracts. Using this cell line, in 1999, we purified a peptide that potently activated GHS-R, and named the GHreleasing peptide ‘‘ghrelin’’. Ghrelin was consisted of 28 amino acids, in which the Ser-3 residue was n-octanoylated. Surprisingly, this n-octanoyl modification at Ser-3 was essential to the activity. We also purified peptides and cloned cDNAs of mammalian and non-mammalian ghrelins. N-termi-

Abstracts nal 10 amino acids are highly homologous, which supports the importance of octanoyl modification for ghrelin’s activity. Ghrelin induced GH release in a dose-dependent manner both in vitro and in vivo. Ghrelin immunoreactive cells in the stomach were found in X/A-like cells, an endocrine cell in stomach. In rat brain, ghrelin immunoreactive neurons were found to be localized in the hypothalamic arcuate nucleus, a region where regulates GH release and food intake. In fact, ICV injection of ghrelin induced potent appetite-stimulating effects in rat. In human studies, i.v. injection of ghrelin elicited a potent GH release and had beneficial hemodynamic effects via decreasing mean arterial pressure and increasing cardiac output without an increase in heart rate. Thus, the occurrence of ghrelin in both stomach and hypothalamus will give a new dimension to the regulation of GH release and feeding, further cardiovascular system and metabolism.

The neuroprotective effects of PACAP on spinal cord injury (SCI) in rats M. Katahira1, K. Yone1, Y. Arishima1, T. Nagamine1, S. Komiya1, S. Iwata2, T. Shimizu2 and A. Miyata2 1 2

Dept. of Orthop. Surg., Kagoshima Univ. Fac. Med., Japan Dept. of Pharmacology, Kagoshima Univ. Fac. Med., Japan

As a nerve degeneration mechanism in acute spinal cord injury (SCI), the bleeding necrosis by the contusion of spinal tissues starts first by direct external force, and it is known that lesion area will be secondarily expanded by biochemical/pathological changes leading to apoptotic cell death. In central nervous system (CNS), there are several reports of neuro-protective actions of PACAP. On the other hand, in spinal cord, there are several reports that PACAP have implications on nociception as a neuromodulator. Recently, it is reported that VIP and PACAP inhibit tumor necrosis factor-a (TNF-a) production in injured spinal cord and in activated microglia. Then we attempted to clarify whether PACAP have neuroprotective effects on SCI. We produced the rat SCI models by extradural static weight-compression at T9. Immediately after SCI, PACAP (10 pmol/10 Al and 100 pmol/10 Al) was medicated intrathecally and the control rats were with artificial CSF only. The spinal cords were removed and studied by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickend labeling (TUNEL) staining, and immunostaining using antibodies against active caspase-3 with time. At 3 days after SCI, in the PACAP group, the presence of TUNEL-positive cells was reduced. Although the analysis of neuroprotective mechanism in SCI is now in progress, this suggests that PACAP may prevent spinal cord from apoptosis and indicates the possibility of clinical application for SCI.

Culture conditions of neuron that affect the neurotoxic effect of LPS and the neuroprotective effect of PACAP T. Kikuta, C. David, M. Li, T. Quebedaux and A. Arimura US – Japan Biomed. Res. Labs., Dept. of Med., Tulane Univ. Health Sci. Ctr., Belle Chasse, LA, USA It is known that minor differences among the culture systems, such as neuron-enriched cultures or neuron/glia co-cultures with different culture media affect the response of neurons to noxious stimuli as well as the neuroprotective action of drugs. We systematically examined the culture conditions that affect the neurotoxic effect of lipopolysaccharide (LPS) and the neuroprotective effect of PACAP. Viable neurons were quantified by the levels of neuron-specific enolase (NSE). Cells were harvested from brain cortex of rats on embryonic day 17. Eighteen different culture conditions were examined that differed in presence or absence of a glial feeder layer, different sera, phenol-red or FrdU. The best neuronal survival was observed in the cultures in DMEM containing 10% FBS for one week without phenolred or FrdU. Progenitors of glial cells grew and differentiated during the culture in 10% FBS. The study was conducted in serum free neurobasal

49

medium. PACAP at 10 13 M and 10 10 M significantly increased neuron survival. The neuroprotective effect of PACAP at 10 10 M was reduced by a cAMP-dependent protein kinase inhibitor, Rp-cAMPS, while the effect at 10 13 M was reduced by an ERK/MAPK inhibitor, PD98059, with a slight attenuation by Rp-cAMP. Indeed, PACAP markedly stimulated ERK-type MAPKs only at femto to picomolar concentrations, while PACAP stimulated adenylate cyclase at high pico to nanomolar concentrations. These findings suggest that the neuroprotection by PACAP at different concentrations is mediated by different mechanisms, possibly via different types of PACAP receptors. Supported by NIH grant R01-NS40373.

Facial nerve recovery in guinea pig and neurotrophic effect of PACAP H. Kimura1,2, E. Ito1, K. Ishikawa1 and M. Kawatani2 1 2

Dept. of Otolaryngol., Akita Univ. Sch. Med., Japan Dept. of Physiol., Akita Univ. Sch. Med., Japan

PACAP (Pituitary adenylate cyclase-activating polypeptide) has neurotrophic effects of neural regeneration and gives protection to the nervous system. We investigated whether PACAP had a neurotrophic effect on peripheral motoneurons and/or if PACAP could facilitate GDNF (glial cell line-derived neurotrophic factor), a neurotrophin, in nerve regeneration. Unilateral transection of the facial nerve was performed in male Hartley guinea pigs and PACAP injected at the site. Saline was substituted as a control. Compound muscle action potentials (CMAP) were recorded to measure the changes of latency. GDNF content in facial target muscle was measured utilizing ELISA. The regenerating site was taken out for histological studies. PACAP hastened the appearance of CMAP and shortened the latency. PACAP increased and prolonged the nerve transection-induced GDNF increase in the facial muscles. The number of myelinated fibers at 4 weeks after the transection was increased. PAC1 receptor and/or VPAC1 receptor were identified in the injury area at 2 days – 4 weeks. These data indicated that PACAP promoted regeneration of the facial nerve.

Cytokines: their receptors and signals—from laboratory to clinic Tadamitsu Kishimoto Osaka University, Osaka, Japan Immune system is essential for protection from microbial infection, but its disorders cause various diseases including allergy, auto-immune diseases, inflammation and several hemopoietic malignancies. Immune system is regulated by a group of proteins with approximately 20 kDa M.W. called cytokines or interleukins, most of which have been molecularly identified. Their receptors and signal transductions have also been largely elucidated. Regulatory T cells can be divided into two groups, Th1 and Th2, according to which cytokines they produce. Overactivation of Th2, which mainly produce IL-4, IL-5 and IL-10 cause allergy and atopic diseases. In contrast, several autoimmune diseases are caused by hyperactivation of Th1 cells, which mainly produce g-interferon. One of the characteristic feasures of cytokines is their functional pleiotropy and redundancy. They have a wide variety of biological activities on various tissues and organs. Moreover, unregulated productions of cytokines, particularly IL-6 or TNFa are involved in various diseases including chronic inflammation, autoimmunity and hemopoietic malignancies. Anti-cytokine therapies, such as anti-TNFa antibody or anti-IL6 receptor antibody have shown dramatic effect on rheumatoid arthritis, Chron’s diseases, Castleman disease, etc.

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Abstracts

Here, I will give a talk on positive and negative regulation of cytokine signals and treatment of inflammatory diseases and hemopoietic malignancies by modulating cytokine signals.

Secretin and autism K. Ko¨ves1, M. Kausz1, D. Reser1 and K. Horva´th2

Cross-talk between PACAP and sonic hedgehog (SHH) pathways in neural stem cells, cerebellar granular progenitor cells and oligodendrocyte progenitors to control cell fate and proliferation V. Lelie`vre1, A. Nicot2, A. Seksenyan1, A. Flores1, C. Ghiani1, J. De Vellis1, E. DiCicco-Bloom2 and J. Waschek1

1

Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary 2 Division of Pediatric Gastroenterology and Nutrition, University of Maryland School of Medicine, Baltimore, MD, USA

1 Department of Psychiatry and The Geffen School of Medicine, University of California at Los Angeles, CA, USA 2 Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA

Secretin is the first discovered gastrointestinal hormone. It is mainly present in enteroendocrine S cells. Secretin was rediscovered in the brain and in endocrine organs; however, its precise localization is still not completely known. Autism was first described and characterized as a behavioral disorder. The major abnormality in the central nervous system is a cerebellar atrophy, and a striking loss or abnormal development of Purkinje cells. The relation between secretin and autism was first demonstrated by one of us. In autistic children, after i.v. administration of secretin, Horva´th and his coworkers observed fivefold higher pancreaticobiliary fluid secretion than in healthy children and, at least in a part of the patients, better mental functions were reported after the secretin test. This observation led us to map a more precise distribution of secretin in the nervous system using an immunohistochemical approach. There were no secretin immunoreactive elements in intact rats; however, after colchicine treatment we found secretin immunoreactivity in several structures: (1) large pyramidal cells in the motor cortex and fibers mainly in the molecular layer; (2) Purkinje cells in the cerebellar cortex and fibers in the superior cerebellar peduncle; (3) cell bodies in the medial, interposed and lateral cerebellar nuclei; (4) superior olivary nucleus; (4) pseudounipolar primary sensory neurons. We have also found interaction between secretin immunoreactive Purkinje cells and substance-P fibers entering the cerebellum. This work was supported by OTKA grant T34429.

PACAP has been shown to exhibit multiple cell-specific growth factor-like actions on various neural precursor populations, including rat oligodendrocyte progenitors (OLP) (Lee et al., 2001), rat and mouse cerebellar granular progenitor (CGP) cells (Nicot et al., 2002) and embryonic day 10.5 mouse hindbrain stem cell (E10.5HB) cultures (Waschek et al., 1998; Lelievre et al., 2002). Because PACAP commonly stimulates protein kinase A activity and PKA activation frequently opposes hedgehog actions, we investigated the interaction of PACAP with SHH on proliferation and on SHH target genes in E10.5HB and CGP cell cultures. SHH stimulated, and PACAP, inhibited DNA synthesis in E10.5HB cultures. PACAP blocked the stimulatory effect of SHH on E10.5HB cultures. In addition, PACAP treatments of E10.5HB modulated expression of SHH target genes observed and quantified by both immunocytochemistry and competitive RT-PCR. In contrast, both SHH and PACAP stimulated the proliferation of OLP. However, PACAP inhibited DNA synthesis in OLP treated with SHH, again in association with decreased expression of SHH target genes. Finally, in CGP cells, PACAP alone had no effect on proliferation, but completely abolished the mitogenic effects of shh. Altogether, the results strongly suggest that PACAP may oppose or/and modulate SHH action in multiple populations of neural progenitors.

Molecular basis of VPAC receptor phenotype M. Laburthe, A. Couvineau and Y.S. Tan

PACAP-mediated intracrine signaling in the testis M. Li1, S. Shioda1,2, H. Funahashi2, C. David1 and A. Arimura1

INSERM U410, Fac. Med. Bichat, 75018 Paris, France

1 US – Japan Biomed. Res. Labs., Tulane Univ. Health Sci. Ctr., Belle Chasse, LA, USA 2 Dept. of Anat., Showa Univ. Sch. of Med., Tokyo, Japan

The hVPAC1 receptor for VIP and PACAP has served as a prototypical class II G protein-coupled receptor (GPCR) for understanding the structure – phenotype relationship of this class of GPCR (1). (i) The N-terminal ectodomain plays a predominant role in VIP binding. A 3D-structure of this domain indicates the presence of a putative binding groove (2) and fits with the existence of 3 SUS bonds between conserved cysteins. Photoaffinity labeling of hVPAC1 receptor with 125I-[Bpa22-VIP] followed by receptor digestions demonstrates physical proximity between VIP and a microdomain within the ectodomain. (ii) Selectivity filters (3) have been characterized. They restrict access to VIP-related peptides (secretin, PHI) and are made of small clusters of amino acids present at the junction between extracellular domains and transmembrane segments (TM). (iii) Cytoplasmic domains required for activation of adenylyl cyclase have been identified (4). Two charged amino acids in intracellular loop 3 and proximal C-terminal tail are crucial. They are strictly conserved in all class II GPCRs. A 3D model of hVPAC1 receptor core (TMs and loops) has been constructed based on the structure of rhodopsin and shows the location of these charged residues. References: (1) Receptors Channels 8, 137 (2002); (2) J. Biol. Chem. 276, 10153 (2001); (3) J. Biol. Chem. 277, 37016 (2002); (4) J. Biol. Chem., on line April 10 (2003).

The PACAP receptor (PAC1-R) is a G-protein-coupled receptor and belongs to the VIP/GHRH receptor family. We found abundant binding sites, which are specific for PACAP with autoradiography and significant levels PAC1-R and a VIP receptor (VPAC2-R) transcripts with RT-PCR in the adult rat testis. A double-staining immunohistochemical study showed co-expression of PACAP and PAC1-R at the same intracellular sites of spermatids, especially in the acrosome. Western blot analysis showed that the majority of PAC1-R-like protein recognized by the rabbit antibody to PAC1-R was found in the cytosolic fraction, followed by the nuclear fraction of the testis. The membrane fraction expressed the least amount of PAC1-R. Unlike the membrane-bound PAC1-Rs, the testicular cytosolic PACAP receptors are coupled to MAPK (ERK1/ERK2), but not to adenylate cyclase. Since no MAPK activation was evoked by a PAC1-Rspecific ligand, maxadilan, in the PAC1-R-depleted testicular cytosol, the cytosolic PAC1-R-like protein is, in fact, coupled to MAPK. When maxadilan or PACAP was added to the spermatid cultures, they stimulated adenylate cyclase with little effect on MAPK. On the other hand, both ligands dramatically activated MAPK but not adenylate cyclase when added to the cytosolic fraction of the spermatids. These findings suggest that PACAP expressed in spermatids directly interact with PAC1-R which is also expressed at the same intracellular sites and activates MAPK. This

Abstracts mode of action could be called ‘‘intracrine’’, in contrast to ‘‘endocrine’’, ‘‘paracrine’’ or ‘‘autocrine’’. This study was supported in part by a grant from Kaken-American Foundation.

Concentration and chromatographic characterization of PACAP in the brains of some lower vertebrates K. Matsuda1, S. Onoue2, K. Kashimoto2, T. Mochizuki3, S. Kikuyama4 and M. Uchiyama1 1

Dept. of Biol., Fac. of Sci., Toyama Univ., Japan 2 ItoHam Foods Inc, Central Res. Inst., Japan 3 Shizuoka Cancer Center Hospital Res. Inst., Japan 4 Dept. of Biol., Sch. of Educ., Waseda Univ., Japan

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 vertebrates. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the brains of some lower vertebrates, such as the bullfrog, stargazer and stingray, using an enzyme immunoassay for PACAP, and HPLC analysis. The concentrations of immunoreactive PACAP in the frog and fish brains were 72 – 340 pmol/g wet weight of tissue. These values were 5 – 20 times as high as those in the mammalian brains. The predominant forms of immunoreactive PACAP in the frog and fish brains were PACAP38 (bullfrog and stargazer) and PACAP44 (stingray). The abundance and broad distribution of immunoreactive PACAP suggest the importance of PACAP in aquatic and semi-aquatic animals.

PACAP enhanced the activity of lumbosacarl parasympathetic preganglionic neurons in the neonatal rat

51

Intercellular communication in the anterior pituitary gland and interleukine-6 (IL-6) C. Mogi1, H. Fukuro1, K. Yokoyama1, A. Takaki2, M. Tomida3 and K. Inoue1 1

Dept. of Reg. Biol., Fac. of Sci., Saitama Univ., Japan Dept. of Integrative Physiol., Grad. Sch. of Med. Sci., Kyushu Univ., Japan 3 Lab. of Carcinogenesis and Cancer Prevention, Saitama Cancer Center Research Institute, Japan 2

To study pituitary cell differentiation, we used the MtT/E cell line derived from a rat pituitary tumor. MtT/E cells were obtained from an estrogen-induced mammotropic tumor, however, they did not produce any pituitary hormones and their growth was not stimulated by estrogen. Because MtT/E cells were positive for Pit-1, which is a common transcription factor for somatotropes, mammotropes and thyrotropes, it was postulated that MtT/E cells might have the potential to produce some hormone. Therefore we treated MtT/E cells with some differentiation factors and found that retinoic acid (RA) induces growth hormone (GH)-producing cells from MtT/E cells. This suggests that MtT/E cells are committed to become somatotrope progenitor cells. Interestingly, MtT/E cells also secrete IL-6 and its secretion is stimulated by the pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), as in the case of agranular folliculo-stellate cells (FS cells) in the anterior pituitary gland. On the other hand, we found that GH and prolactin (PRL) secretion from somatomammotropic cell line (MtT/SM), were strongly inhibited by cytokines such as IL-6, leukaemia inhibitory factor (LIF). These results suggest that cytokines play important roles for regulation of endocrine cell differentiation in the anterior pituitary gland, i.e., PACAP/ VIP stimulates IL-6 secretion from FS cells or endocrine progenitor cells and IL-6 regulates endocrine cells differentiation.

The development of VIP-chemotherapeutic conjugates T.W. Moody1, G.. Czerwinski2, N.I. Tarasova2 and C.J. Michejda2

1

2

1

Akira Miura , William C. de Groat and Masahito Kawatani

1

DHEW, NIH, NCI Office of the Director, CCR, Bethesda, MD, USA Macromolecular Structure Lab. Advance BioScience Lab., NCI, Frederick, MD, USA

2 1

Department of Physiology, School of Medicine, Akita University, Akita 010-8543, Japan 2 Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5 – 11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 Al of 4% solution) injected into the intraperitoneal space before experiments. Bath application of PACAP (20 nM) unmasked or increased spontaneous firing of PGN in the presence of CNQX (5 AM), APV (50 AM), strychnine (1 AM) and bicuculline (10 AM). In phasic and tonic PGN, PACAP increased by 109 – 120% ( P < 0.05) the frequency of firing elicited by depolarizing current pulses. PACAP also increased the number of spikes and decreased the latency for firing followed by a hyperpolarizing current pulse. PACAP decreased the threshold for spike generation (from 37.3 F 1.8 to 42.7 F 2.2 mV), and shortened spike duration at the half amplitude (from 2.2 F 0.3 to 1.9 F 0.2 ms). The duration of spike after-hyperpolarization was decreased (from 202.5 F 25.3 to 112.7 F 24.3 ms) by PACAP, but the amplitude was not changed. PACAP suppressed a transient outward current, which was also suppressed by 4-aminopyridine (5 mM). In the presence of TTX, PACAP increased the amplitude of spontaneous EPSPs and also increased the frequency of spontaneous EPSPs. These results suggest that PACAP may modulate the excitability of lumbosacral parasympathetic preganglionic neurons.

VIP is an autocrine growth factor for some lung cancer cells. Here the effects of VIP-ellipticine (E) conjugates were investigated on lung cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited specific 125I-VIP binding to NCI-H1299 cells with IC50 values of 0.7 and 0.3 AM. Both VIPALALA-E and VIP-LALA-E caused cAMP elevation and increased c-fos mRNA in NCI-H1299 cells suggesting that both VIP-E conjugates function as agonists. Radiolabeled VIP-LALA-E was internalized at 37 jC and delivered cytotoxic E into NCI-H1299 cells. VIP-LALA-E decreased NCIH1299 colony formation in vitro. VIP-LALA-E reduced 3H-leucine and 3Hthymidine uptake into NCI-H1299 cells, decreasing protein and DNA synthesis, respectively. The results indicate that VIP-E conjugates are cytotoxic for lung cancer cells.

Effect of PACAP on POMC and NPY mRNA expression in the rat hypothalamus L. Mounien1, P. Bizet1, I. Boutelet1, A. Fournier2, H. Vaudry1 and S. Je´gou1 1 Eur. Inst. Pept. Res., Lab. Cell. Mol. Neuroendocrinol., INSERM U413, Univ. Rouen, France 2 INRS, Univ. Quebec, Montreal, Canada

Central administration of PACAP inhibits food consumption in rat while disruption of the type II PACAP/VIP receptor (VPAC2-R) gene increases the

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Abstracts

basal metabolic rate in mice. The arcuate nucleus (AN) of the rat hypothalamus contains a dense population of proopiomelanocortin (POMC) and neuropeptide Y (NPY) cell bodies that exert opposite actions in energy homeostasis. In addition, the AN exhibits a high density of type I PACAP receptor (PAC1-R) and VPAC2-R. These observations led us to investigate the possible effect of PACAP on POMC and NPY neurons. Rats received an i.c.v. injection of PACAP (1 or 5 Ag) or VIP (1 Ag), and the levels of POMC and NPY mRNAs were quantified by in situ hybridization. At doses of 1 and 5 Ag, PACAP provoked a 48% and 91% increase in NPY mRNA expression, and a 47% decrease (for both the 1- and 5-Ag doses) in POMC mRNA expression. Injection of VIP, a VPAC2-R agonist, mimicked the effect of PACAP on POMC ( 48%) and NPY ( + 45%) mRNA expression. The present data suggest that PACAP, acting through VPAC2-R, regulates energy expenditure by modulating the activity of POMC and NPY neurons. This work was supported by INSERM (U413), an INSERM-FRSQ exchange program, and the Conseil Re´gional de Haute-Normandie.

Low concentration of PACAP induces differentiation of mouse neural stem cells into astrocytes via Gq pathway S. Nakajo1, F. Ohno1, H. Sekihara1, T. Hirabayashi1, S. Shioda2 and K. Nakaya1 1 2

Lab. Biol. Chem., Showa Univ. Sch. Pharmaceut. Sci., Japan Dept. of Anatomy, Showa Univ. Sch. Med., Showa, Japan

We have found that low concentration (2 nM) of PACAP induces differentiation of mouse neural stem cells to astrocytes. Although the differentiation was not affected by cAMP analogues such as dibutylic cAMP or 8Br-cAMP, or specific competitive inhibitor for protein kinase A, Rp-cAMP, it was mimicked by PMA but not by 4a-PMA. Expression of PACAP receptors (PAC1) in neural stem cells was confirmed by both the methods of RT-PCR and immunoblot with an affinity-purified antibody. PACAP selective antagonist, PACAP6 – 38 showed inhibitory effect on the differentiation by PACAP in neural stem cells. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, which in turn transmit the signal to PAC1-coupled Gprotein Gq but not to Gs and may thereby play a crucial role in differentiation of neural stem cells to astrocytes.

Isolation and purification of gastric ECL cells D.S. Oh, P.M. Germano and J.R. Pisegna VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA Introduction: The enterochromaffin-like (ECL) cells of the gastric fundus have been shown to express PAC1. A fluorescent PACAP ligand could be useful to isolate ECL cells from the rat by taking advantage of our knowledge of the expression of PAC1 by these cells. Aims: To develop a technique to isolate rat ECL cells to near-purity using fluorescence-activated cell sorting (FACS) using a fluorescent ligand. Methods: Fundic tissue, extracted from rats, was digested using a combination of hyaluronidase/collagenase B. Subsequently; the isolated gastric cells were subjected to FACS. The sorted cells were identified as ECL cells via immunohistochemical analysis. Furthermore, to validate the presence of PAC1 on ECL cells, confocal microscopy was utilized to verify the surface expression of PAC1 by using Fluor-PACAP38. The viability of ECL cells was determined by MTT Elisa assay. Results: Approximately 1.5  106 cells were obtained from the stomachs of two rats using enzymatic treatment. Between 7.2% and 8.5% of the total fundic cells sorted by the FACS machine were collected. The final purity of the isolated ECL cells was observed to be greater than 99% with a yield of 5.0 – 9.0  105 cells. FACS putatively isolated ECL cells based on two criteria: their relatively small size and their ability to bind the fluorescent ligand FluorPACAP38. The uptake of MTT by unstimulated ECL cells demonstrated their viability. Conclusions: The collected ECL cells composed about 4 – 6% of the approximate 1.4 – 1.6  107 cells available before sorting, consistent with the published data. With the ability to isolate such a pure collection of ECL cells, further microarray analyses employing ECL mRNA extracts will enable the deciphering of gene expression under various conditions.

Pharmacological usefulness of dry powder inhaler of a novel vasoactive intestinal peptide (VIP) analogue as anti-asthma agent Y. Ohmori1, S. Yamada1, R. Kimura1, S. Onoue2, A. Matsumoto2, K. Endo2, T. Iwanaga3 and K. Kashimoto2 1

Effects of aging, amyloid B protein, and lipopolysaccharide on the transport of PACAP across the blood –brain barrier N. Nonaka

1,2,3

, W.A. Banks3 and S. Shioda2

1

Dept. of Oral Anatomy, Showa Univ. Sch. Dent., Japan Dept. of Anatomy, Showa Univ. Sch. Med., Showa, Japan 3 Dept.of Internal Medicine, Veterans Affairs Medical Center, St. Louis Univ. Sch. Med., St. Louis, MO, USA 2

The blood – brain barrier (BBB) controls the exchange of peptides and regulatory proteins between the central nervous system and the blood. We measured the transport rate of radioactively labeled pituitary adenylate cyclase-activating polypeptide (PACAP) from blood into whole brain and into 11 brain regions in young ICR, young SAMP8 (a model of Alzheimer’s disease) and aged SAMP8 mice. We found that I-PACAP crossed the BBB faster at the hypothalamus and the hippocampus in all three groups. Slower transport rates into the whole brain, the olfactory bulb, the hypothalamus, and the hippocampus for aged SAMP8 mice was likely related to differences both from strain and the overexpression of amyloid h protein with aging. In addition, we found the transport of I-PACAP across the BBB to be decreased after the injection of LPS. These results show that the PACAP BBB transporter is regulated by pathophysiologic events.

Sch. Pharm. Sci. and COE21, Univ. of Shizuoka, Japan Health Sci. Div., Itoham Foods Inc, Japan 3 Grad. Sch. Med., Hokkaido Univ., Japan 2

VIP has a wide range of biological actions such as vasodilation, bronchodilation and anti-inflammatory effect. Currently, the deficiency of VIP in the respiratory system is suggested to be one of etiopathogenic factors in the pulmonary diseases including asthma. Because of its bronchodilating and anti-inflammatory effects, VIP has been suggested to be a promising agent to treat pulmonary diseases. However, the therapeutic application of VIP is largely limited by the rapid enzymatic degradation in addition to the systemic adverse effects due to the wide distribution of VIP receptors. To overcome these problems, we have recently developed dry powder inhaler of novel VIP analogue (IK312532) having high stability against the enzymatic digestion, directed toward an effective therapeutic agent of asthma. IK312532 relaxed potently the histamine-induced contraction of isolated guinea-pig trachea with longer duration than VIP did. Compared with VIP, IK312532 exhibited significantly higher binding affinity to specific [125I]VIP binding sites in the rat lung homogenate. The intratracheal administration of IK312532 as dry powder inhaler was shown to bind significantly to VIP receptors in the rat lung and to suppress markedly the infiltration of granulocyte (eosinophil, neutrophil) in the rat tracheal mucosa after the antigen inhalation. In conclusion, the present study has shown that dry powder inhaler of IK312532 may be a pharmacologically useful drug delivery system for the therapy of pulmonary diseases such as asthma.

Abstracts

Neuroprotective effect of PACAP38 through IL-6 after focal ischemia in mouse H. Ohtaki1,5, K. Dohi2, L. Yin1, T. Nakamachi1, A. Takaki3, S. Nakajo4 and S. Shioda1,5 1

Dept. of Anatomy, Showa Univ. Sch. Med., Japan Emergency and Clinical Care Medicine, Showa Univ. Sch. Med., Japan 3 Dept of Integrative Physiol., Graduate Sch. of Medical Sci., Kyushu Univ., Japan 4 Lab. Biol. Chem., Showa Univ. Sch. Pharm. Sci., Japan 5 CREST of JST, Japan 2

We have showed previously that infusion of PACAP38 protects ischemic delayed neuronal cell death (DNCD) in rat hippocampus. Moreover, microinfusion of PACAP38 after global ischemia immediately increases IL-6 concentration in the cerebrospinal fluid after brain ischemia. However, it is unknown in detail whether PACAP plays a neuroprotective role through IL-6 or not. Therefore, the purpose of the present study is to clarify the neuroprotective role of PACAP after brain ischemia by using the wild-type (wild) and IL-6 KO mice. The mice were subjected to 1h transient middle cerebral artery occlusion (tMCAO) using the intrasuture methods. PACAP38 (16 pmol/0.5 Al/h) or vehicle (0.9% saline containinig 0.1% BSA) was infused intravenously with osmotic pump followed by bolus injection (5 nmol/kg). Twenty-four hours after ischemia, PACAPtreated wild mice decreased the infarct volume and the neurological score was lower than the control. However, there were no neuroprotective effects of PACAP treated IL-6 KO mice. To determine the neuroprotective mechanism of PACAP, the PAC1-R immunostaining was performed after tMCAO. PAC1-R immunoreactivity was expressed in neurons of the cortical 3rd layer in the control mice. The many PAC1-R expressing neurons appeared in the ipsilateral hemisphere after tMCAO. The correlation between IL-6 release and PAC1-R expression is more studied in detail.

Vasoactive intestinal peptide protects rat alveolar L2 cell from the cytotoxicity of cigarette smoke Satomi Onoue1,2, Kosuke Endo1, Yuki Ohmori3, Shizuo Yamada3, Ryohei Kimura3, Takehiko Yajima2 and Kazuhisa Kashimoto1 1

Health Sci. Div., Itoham Foods Inc., Ibaraki 302-0104, Japan 2 Dept. Anal. Chem., Sch. Pharm. Sci. Toho Univ., Chiba 274-8510, Japan 3 Dept. Biopharm., Sch. Pharm. Sci. and COE21, Univ. Shizuoka, Shizuoka 422-8526, Japan Vasoactive intestinal peptide (VIP) acts as neurotransmitter in numerous biological responses. In the present study, we investigated the effect of this neuropeptide on the cytotoxicity of cigarette smoke, major causative factor of chronic obstructive pulmonary disease (COPD), in rat alveolar L2 cells. Dulbecco’s modified eagle medium (25 ml) was bubbled well with the smoke from two sticks of research cigarette (2R4F) in attempt to prepare the cigarette smoke extracts (CSE). The CSE at the concentration of 0.1% or higher displayed the significant apoptotic death of L2 cells, as evidenced by the significant increase of LDH release, decrease of WST-8 reduction, and DNA fragmentation. In addition, the CSE-induced cytotoxicity was attenuated by the addition of inhibitors for caspases (Z-VAD-FMK, Ac-DEVDCHO) and matrix metalloproteinase (MMP; GM6001), indicating that the caspases and MMP pathways may be involved in the cytotoxicity of CSE in L2 cells. In L2 cells, the RT-PCR experiment displayed the dominant expression of mRNA for VPAC2 receptor and VIP stimulated the adenylate cyclase in a concentration-dependent manner. Interestingly, the addition of VIP at the concentration of 10 10 M or higher in L2 cells with CSE (0.25%) resulted in a significant attenuation of cell death with the deactivation of the CSE-stimulated caspase-3 and MMP activity. In conclusion, it has been suggested that VIP is applicable to the clinical treatment for COPD.

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PACAP enhances the hyperpolarization activated cationic conductance, Ih, in dissociated guinea pig cardiac neurons R. Parsons, K. Barstow and L. Merriam Department of Anatomy and Neurobiology, University of Vermont, Burlington, VT 05405, USA PACAP peptides are co-localized with acetylcholine in preganglionic parasympathetic fibers innervating the guinea pig intracardiac ganglia. The cardiac neurons express PAC1 receptors, predominantly the very short variant with neither hip nor hop cassettes. PACAP27 increases excitability of the cardiac neurons such that the number of action potentials produced by a depolarizing current pulse or following anodal break is increased. The present study used perforated patch clamp techniques to test whether a PACAP27-enhanced activation of Ih could contribute to the increased excitability. The effects of 1 – 100 nM PACAP27 on Ih were determined during hyperpolarizing voltage steps (1.5 s) from a holding potential of 50 mV to voltages between 60 and 130 mV. PACAP27 increased the amplitude and rate of development of the inward current profile typical of Ih. PACAP27 produced a concentration dependent positive shift (4.5 – 6.7 mV, 1 – 100 nM) in the voltage dependence of activation determined from normalized tail current amplitudes fitted to a Boltzmann function. The effect of PACAP27 was eliminated by the Ih inhibitors CsCl and ZD7288. The adenylyl cyclase activator forskolin (10 AM) produced a similar shift in the voltage dependence of Ih activation. We propose that PACAP27 enhances Ih, an effect mediated by PAC1 receptor activation of adenylyl cyclase and generation of c-AMP, and that this contributes to the PACAP27-induced increase in excitability. Supported by NIH grant HL65481.

Bone morphogenetic proteins (BMPs) regulate PACAP expression in sympathetic neurons K.A. Pavelock, B.M. Girard, K.N. Dozark, K.M. Braas and V. May Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA The principal postganglionic neurons of the rat sympathetic superior cervical ganglion (SCG) express preferentially the PAC1(short)-HOP1 receptor splice variant coupled to multiple intracellular signaling cascades. PACAP synthesized by sympathetic pre-ganglionic projection neurons to the SCG have been implicated to modulate sympathetic function; however, low endogenous levels of PACAP expression in SCG postganglionic neurons are augmented dramatically by neuronal depolarization or injury, but the regulators of PACAP expression have not been well established. BMPs belong to the TGFh superfamily of target tissue factors, which are retrogradely transported in neurons to alter gene expression. Our current studies examine the roles of the Gbb/60A BMP subclass, including BMP5, 6 and 7, in neuronal dendritic fiber outgrowth and differentiation. BMP6 diminished significantly PACAP transcript expression in primary cultured sympathetic neurons. Using semiquantitative RT-PCR, the effects of BMP6 were rapid, potent (nM IC50), and sustaining. Parallel microarray analyses of BMP-treated sympathetic cultures support these results. We have identified Gbb/ 60A BMP proteins as negative regulators of PACAP expression that may function to modulate SCG PACAP levels in vivo. Conversely, disruption of BMP availability may contribute to augmented PACAP expression in neuronal repair/regeneration after injury. Supported by NIH HD27468, NS37179, P30CA22435 and AHA 0120367T.

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Abstracts

Vasoactive intestinal peptide: a new drug for treatment of primary pulmonary hypertension Ventzislav Petkov1, Wilhelm Mosgoeller2, Rolf Ziesche1, Markus Raderer3, Leopold Stiebellehner1, Karin Vonbank1, Georg-Christian Funk1, Gerhard Hamilton5, Clemens Novotny4, Bernhard Burian1 and Lutz-Henning Block1 1

Department of Internal Medicine IV, Division of Pulmonary Medicine, University of Vienna, Medical School, Vienna, Austria 2 Institute of Cancer Research, University of Vienna, Vienna, Austria 3 Department of Internal Medicine IV, Division of Oncology, University of Vienna, Medical School, Vienna, Austria 4 Department of Nuclear Medicine, University of Vienna, Medical School, Vienna, Austria 5 Ludwig Boltzmann Institute of Clinical Oncology, KH Lainz, Vienna, Austria Background: Primary pulmonary hypertension is a fatal disease causing progressive right heart failure within 3 years after diagnosis. Vasoactive intestinal peptide (VIP) is a potent systemic and pulmonary vasodilator. We hypothesized that VIP might be involved in the pathogenesis of primary pulmonary hypertension (PPH) and might be used for its treatment. Methods: Our rationale is based on the finding of a deficiency of the peptide in serum and lung tissue of patients with primary pulmonary hypertension as evidenced by radioimmunoassy and immunohistochemistry. The relevance of this finding is underlined by an upregulation of corresponding receptor sites as shown by Northern blotting, Western blotting, and immunological techniques. We measured VIP serum concentration in PPH patients and healthy individuals. Receptor expression and 125 I-VIP binding was determined in pulmonary artery smooth muscle cells. In a prospective, intraindividual study eight PPH patients inhaled aerosolized VIP (200 Ag daily) for 6 months. Results: In PPH, VIP serum levels were at 10 pg/ml compared to 42.4 F 17.1 pg/ml in normals. VIP receptor-expression and specific binding was increased in PPH. Three months of VIP inhalation decreased mean pulmonary artery pressure by 13 mm Hg from 59 F 8 to 46 F 7 mm Hg ( p < 0.01). Cardiac output increased by 1.7 l/min from 4.7 F 2.0 to 6.4 F 1.6 l/min ( p < 0.01). Pulmonary vascular resistance decreased from 1009 F 475 to 586 F 165 dyn s 1
cm 5 ( p < 0.01). Mixed venous oxygen saturation increased from 58 F 7% to 63 F 7% ( p < 0.01). The 6-min walk distance increased by 113 m from 296 F 138 to 409 F 102 m ( p < 0.01) after 12 weeks and by 129 m from 296 F 138 to 425 F 107 m ( p < 0.01) after 24 weeks VIP treatment. Conclusions: Our data provide ‘‘proof of concept’’ for further investigation of vasoactive intestinal peptide and it’s role in primary pulmonary hypertension.

Toward the isolation and characterization of the VIP receptor(s) that mediate neuronal survival I. Pilzer1, M. Zusev1, R.A. Steingart1, D. Dangoor1, S. Rubinraut2, M. Fridkin2, D.E. Brenneman3 and I. Gozes1 1

Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Organic Chemistry, Weizmann Institute, Israel 3 SDMP, LDN, NICHD, NIH, Bethesda, MD, USA An antisense oligodeoxynucleotide specific for the HOP2 splice variant of the PAC1 receptor kills neurons in cell culture, and reduces VIP binding to glial cells. These results implicate HOP2 in VIP-mediated neuroprotection (J. Mol. Neurosci. 1997 Dec.;9(3): 211 – 22). Furthermore, the newly cloned activity-dependent neuroprotective protein (ADNP) was shown to mediate, in part, VIP-induced neuroprotection. The aim of the present work was to: (1) examine the affinity of the cloned HOP2 receptor to VIP and (2)

identify the VIP receptor associated with increases in ADNP expression. The PAC1 cDNA was PCR-cloned from rat cerebral astrocytes and genetically manipulated to obtain the HOP2 splice variant. It was then inserted into an expression vector and transfected into COS-7 cells that were used for 125I-VIP binding assays. Results showed, for the first time, that VIP bound the cloned HOP2 PAC1 splice variant. Stearyl-Nle17Neurotensin6 – 11 VIP7 – 28(SNH), that potently kills neurons, was found to bind HOP2 as well. In parallel experiments, using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. Thus, VPAC2 and HOP2 are implicated here in neuronal survival and it is hypothesized that the ratio of the different VIP/PACAP receptor expression levels in a given glial/neuronal cell may influence the outcome of VIP/ PACAP neuroprotective effects. Support: ISOA, Gildor Chair, BSF, Neufeld award, ISF and Allon Therapeutics.

Effects of PACAP treatment in a rat model of Parkinson’s disease D. Regl} odi, A. Lubics, A. Tama´s, L. Szalontay and I. Lengva´ri Department of Anatomy, Pe´cs University, Hungary PACAP has numerous neurotrophic and neuroprotective effects both in vitro and in vivo. Among others, it has been shown to protect mesencephalic neurons against 6-hydroxydopamine (6-OHDA)-induced cell death. The aim of the present study was to investigate whether PACAP has neuroprotective effects also in an in vivo model of Parkinson’s disease. The substantia nigra pars compacta was damaged by unilateral administration of 6-OHDA. Animals received either physiological saline only (normal control group), 6-OHDA and physiological saline, or 6-OHDA and different doses of PACAP38. This model of Parkinson‘s disease produces several signs of motor assymetry. One and 10 days after the operation, rats were video-recorded in an open-field for 15 min to test their motor behavior. Also, other neurological signs such as limb flexion, balance, walking and sensory functions were tested. After completing the behavioral testings, animals were sacrificed and the dopaminergic cells in the substantia nigra were investigated by tyrosine-hydroxylase immunohistochemistry. Our findings show that both 6-OHDA and PACAP-treated groups showed severe hypokinesia and assymetries 1 day after the lesion as compared to the normal control group. However, PACAP-treated animals performed significantly better, especially 10 days after the operation, which shows that they recovered better. The histological examination showed severe loss of dopaminergic cells in the 6-OHDA treated animals as compared to the control group and the uninjured side. The cell loss was significantly less in the PACAP-treated group. These findings suggest that PACAP has a protective effect in a rat model of Parkinson’s disease. Supported by OTKA 034491 and MTA.

Possible factors for the neuroprotective effect of PACAP in focal cerebral ischemia D. Regl} odi1, Zs. Fa´bia´n2, A. Tama´s1, J. Szebere´nyi2, T. Alexy4, K. To´th4, Zs. Ma´rton4, B. Borsiczky3 and I. Lengva´ri1 1

Dept. of Anatomy, Pe´cs University Medical Faculty, Hungary Dept. of Biology, Pe´cs University Medical Faculty, Hungary 3 Dept. of Experimental Surgery, Pe´cs University Medical Faculty, Hungary 4 First Dept. of Medicine, Division of Cardiology, Pe´cs University Medical Faculty, Hungary 2

In previous studies, we showed that PACAP treatment reduced the infarct size in a rat model of focal cerebral ischemia. The aim of the present study was to investigate some of the possible factors included in the protective mechanism of PACAP. The antiapoptotic effect of PACAP has

Abstracts been shown in various neuronal cultures by others. In the present study, we investigated the effect of PACAP in anizomycine-induced apoptosis of PC12 cells. We found that PACAP prevented apoptosis in a range of 1 AM – 10 pM, which was diminished in a PKA-deficient culture showing that PKA is necessary for the antiapoptotic effect of PACAP. This effect was also studied in vivo, in a rat stroke model. The number of apoptotic cells in sections of infarcted brain areas stained by TUNEL technique was significantly less in the PACAP-treated group than in control animals, especially in the infarct core. Platelet aggregation was studied by inducing aggregation by ADP, collagen or epinephrine in human platelet-rich plasma. PACAP in the range of 1 AM – 10 pM had no effect on platelet aggregation. In similar concentrations, PACAP showed no effects on the oxidative damage of red blood cells or on the luminol-dependent reactive oxygen intermedier production of leukocytes. Our conclusion is that among the possible factors studied, antioxidant effects and effects on platelet aggregation play no role in the neuroprotective actions of PACAP, but the antiapoptotic affect might play a significant role in reducing the infarct size. Supported by OTKA 034491 and MTA.

VIP and PAGAP via G-protein coupled receptors are potent inducers of mouse embryonic stem cell neuronal differentiation

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Experiments in vitro, in isolated organs and in vivo demonstrate that VIP can reduce or prevent apoptosis in lung and neuronal cells, and promote survival of cells, organs and whole animals. Injury models so far tested include oxidant stress (induced by paraquat or xanthine oxidase) and excitotoxicity (induced by glutamate agonist N-methyl-D-aspartate) in lungs; trophic withdrawal in hippocampal progenitor cells; and glutamate toxicity in neuronal-like pheochromocytoma (PC-12) cells. In these models, VIP inhibited apoptosis by one or more of three mechanisms: inhibition of caspase activation, upregulation of the antiapoptotic protein bcl2 and suppression of cytochrome-c translocation. In the in vivo studies, VIP prolonged survival of rats and mice subjected to shock due to sepsis or massive hemorrhage. These data suggest that VIP is of potential benefit in modulating programmed cell death in vital organs and in promoting overall survival in a number of life-threatening conditions.

Pituitary adenylate cyclase-activating polypeptide (PACAP) protects ganglion cell death against cutting of optic nerve in the rat retina Seki Tamotsu1, Izumi Sachiko2, Shioda Seiji3 and Arimura Akida4 1

Kozawa Eye Hospital Eye Research Center, Ibaraki, Japan Department of Ophthalmology, Showa University, School of Medicine, Japan 3 Department of Anatomy, Showa University, School of Medicine 4 US – Japan Biomedical Research Laboratories, Tulane University, Hebert Center, Belle Chasse, LA, USA 2

W. Roste`ne,1 B.J. Gonzalez,2 A. Lombet,1 P. Gressens,3 H. Vaudry2 and M. Cazillis3 1

INSERM E0350, Paris, France INSERM U413, Mt. St. Aignan, France 3 INSERM E9935, Paris, France 2

Mouse embryonic stem cells (ES cells) are totipotent cells derived from 3.5-day blastocysts. VIP and PACAP are known to affect neuronal differentiation but possible effects at the earliest times of ontogeny have not been described. We thus checked whether these peptides may have any effect on ES cells. ES cells from 129 SV/EV mice were allowed to aggregate in embryoid bodies (EB) in presence or not of VIP or PACAP for one week. Spontaneous differentiation of ES cells in a neuronal-like phenotype represented around 10%, 15% were oligodendrocytes and no astrocytes were found. VIP dramatically increased the number of EBderived cells (95%) expressing neuronal markers with no change in glial markers. It correlates with an increase in processes number and length. PACAP was more potent than VIP to induce such differentiation. 45% of the neuronal-like cells (Tuj1+) expressed tyrosine hydroxylase and 40% GABA. Binding competition of 125IPACAP27 with VIP and PACAP as well as RT-PCR analysis revealed the presence of VPAC receptor subtypes and PAC1 receptor on ES cells whereas only a functional PAC1 was found on EB-derived cells as observed during brain development. Similarly, both VIP and PACAP increased cAMP and Ca2+ in ES cells whereas only PACAP was active on EBs. These original data suggest that functional GPcR neuropeptide receptors can be present at the first stages of development and thus may play a role in the differentiation of ES cells into neural precursors and progenitor neurons. It opens an exciting new field of research for neuropeptide/neurotransmitter regulation of tissue ontogenesis.

VIP as an anti-apoptotic, pro-survival agent

Purpose: We have reported already that pituitary adenylate cyclaseactivating polypeptide (PACAP) prevents from retinal ganglion cell death using ischemia – repefusion model by a rat intra-ocular hypertension. We examined effects of prevention from delayed neuronal cell death by PACAP using an optic nerve cutting model of the rat. Methods: We made a dissection of optic nerve cutting model and the 3 Al of the control medium (vehicle) or PACAP was injected into the vitreous body at once. At seventh day after the treatment, eyes were enucleated and posterior segments were fixed immediately. The retinal ganglion cell numbers were counted on the light microscope. The data between PACAP groups and vehicle group were examined using Dunnet (two-tailed). Results: The number of survival ganglion cells decreased apparently in the injected vehicle group compared with the no cutting treatment. In the group that injected 10 pmol/3 Al and 100 pmol/3 Al with PACAP, the numbers of survival ganglion cells were statistically larger than the group of the vehicle injected into the vitreous body in this model. Conclusions: These findings suggest that PACAP protects ganglion cell death from insult in cutting optic nerve.

Functional aspects of PACAP as deduced by genetic and other manipulations Nancy M. Sherwood1, Nola M. Erhardt1, Petra Vencova1, Kaaren Gibbs1, Sarah L. Gray1, Kevin J. Cummings1 and Frank R. Jirik2 1 2

Sami I. Said, Kathleen G. Dickman, Collin Brathwaite and Francis J. Antonawich State University of New York at Stony Brook and VA Medical Center, Northport, NY, USA Apoptosis, or programmed cell death, is a leading mechanism of cell death and organ dysfunction, and one that can be targeted for therapeutic intervention.

Dept. of Biology, University of Victoria, Canada Dept. Biochem. and Mol. Biology, University of Calgary, Calgary, Canada

PACAP is expressed as a 27 or 38 amino acid peptide in all vertebrates studied to date. Diverse functions have been proposed for PACAP, but the essential ones are uncertain. We have examined a number of functions using genetic manipulations and other methods. We conclude that PACAP is an essential hormone needed during certain challenges or stresses. We found that during early embryonic brain development, PACAP is important in maintaining a high level of neuroblast proliferation and in preventing apoptosis as shown by

56

Abstracts

neuronal cell culture and FACS analysis. Also, during early postnatal development, protection of core body temperature by PACAP acting on the sympathetic nervous system via brown fat is essential as shown in a PACAP-gene-knockout mouse. PACAP is crucial in mice at low temperatures (21 C) in maintaining lipid distribution. Long-term treatment with PACAP shows that excess PACAP did not alter carbohydrate metabolism, insulin secretion, lipid distribution or growth. This suggests that the primary function of PACAP is not critical for normal homeostasis, but is essential under certain stress conditions. To pinpoint the location of PACAP that underlies essential functions, conditional knockouts have been prepared.

the deficit in PPI as well as hyperactivity and jumping behavior. A selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine (2.5 – 10 mg/kg) also effectively suppressed the hyperactivity and jumping behavior. A 5-HT1A receptor agonist 8-OH-DPAT (0.5 mg/kg) significantly lowered locomotor activity and rectal temperature in wild-type mice, while it had only a small effect in PACAP-KO. These results suggest the involvement of monoaminergic dysfunctions in phenotypic changes of PACAP-KO.

Effects of intrathecal PACAP injection on nociceptive transmission in mice

H. Sugahara1,2, T. Shimizu1, S. Iwata1, K. Yamada2, N. Mori3 and A. Miyata1

T. Shimizu1, M. Katahira2, H. Sugahara3, K. Inoue1 and A. Miyata1

Involvement of neural restrictive silencer (NRS) in the regulatory mechanism of PACAP gene expression

1

Dept. Pharmacology, Kagoshima Univ., Kagoshima 890-8520, Japan Dept. Clinical Pharmacy, Fac. Med., Kagoshima Univ., Kagoshima 890-8520, Japan 3 Dept. Mol. Genetics, Nat. Inst. Longevity Science, Aichi 474-5822, Japan 2

1

Dept. Pharmacol., Fac. Med., Kagoshima Univ., Japan Orthop. Surg., Fac. Med., Kagoshima Univ., Japan 3 Clin. Pharm., Fac. Med., Kagoshima Univ., Japan 2

PACAP immunoreactive projections are observed in the superficial layers of the dorsal horn suggesting a role for PACAP related to nociceptive transmission and modulation. Controversial effects were reported on whether PACAP is nociceptive or antinociceptive. We examined nociceptive transmission of PACAP in the mouse spinal cord. Agents were intrathecally injected to male ddY mice. Aversive responses (licking and scratching) were counted every 1 min. The injection of substance P (SP: 6 – 100 pmol) immediately displayed aversive responses and disappeared within 1 – 2 min. While PACAP38 (100 pmol) expressed continuous aversive responses for 1 – 2 h following long latency (about 130 s). During the latency, PACAP produced an increase in tail flick latency (thermal nociception) and mechanical nociceptive threshold for tail pinch. Furthermore, the PACAP or maxadilan (25 pmol: PAC1-R agonist) displayed hyperalgesia (enhanced 9 pmol SP-induced responses) on the next day, but did not display hyperalgesia for mechanical nociception (tail pinch). The hyperalgesia disappeared after max. d.4 (PAC1-R antagonist) coinjection. These results suggest diverse effects of PACAP on nociception, such as an analgesic role in early time of the injection and subsequently lasting algesia, further hyperalgesia for SP-induced aversive responses.

Altered behavioral response to monoaminergic drugs in mice lacking PACAP N. Shintani1, K. Tanaka1, H. Hashimoto1 and A. Baba1,2 1 Lab. of Mol. Neuropharmacol., Grad. Sch. Pharmaceut. Sci., Osaka Univ., Osaka, Japan 2 Lab. of Mol. Pharmacol., Grad. Sch. Med., Osaka Univ., Osaka, Japan

PACAP is a pleiotropic neuropeptide that belongs to the secretin/ glucagon/VIP family. Previously we undertook the detailed analysis of 5V upstream region of mouse PACAP gene and identified the neural restrictive silencer (NRS) like elements (NRSLE1 and 2) in apploximately 1.7 – 1.9 kbp upstream from the translation start site. In PC12 cells, nerve growth factor (NGF) induces neurite outgrowth and augments PACAP mRNA. In order to clarify the contribution of NRS to PACAP gene expression in PC12 cells, we analyzed the silencer activity by using a SV40-luciferase reporter construct containing NRSLE1 or 2 of mouse PACAP gene. In untreated PC12 cells, both the NRS like elements (NRSLE1 and 2) equally and significantly repressed the SV40 promoter activity. In PC12 cells differentiated with NGF; however, these repressions were attenuated completely. In order to analyze the specific binding protein of NRSLE1 and 2, we performed electrophoretic mobility shift (EMS) assays on nuclear extracts from PC12 cells using the NRSLE1 or 2 containing oligonucleotide as a probe. Either NRSLE1 or 2 specific complexes were competitively inhibited with the NRS of rat type II sodium channel. Further in RT-PCR analysis of differentiated PC12 cells, the mRNA level of REST4/NRnV, the dominant negative form of NRS binding factor (NRSF), significantly increased as compared with that of NRSF itself. These suggest that the NRS might be involved in the regulatory mechanism of PACAP gene expression in PC12 cells.

Upregulation of PACAP receptor in the reactive astrocytes in mice neocortex after brain injury R. Suzuki1,2, S. Arata3, S. Nakajo4, S. Kikuyama2 and S. Shioda1,5 1

Dept. of Anatomy, Showa Univ. Sch. Med., Japan Dept. of Biol., Sch. Educ., Waseda Univ., Japan 3 Ctr. for Biotech., Showa Univ., Japan 4 Lab. of Biol. Chem., Showa Univ. Sch. Pharm. Sci., Japan 5 CREST of JST, Japan 2

We have previously reported the generation of mice lacking PACAP (PACAP-KO), which exhibit altered psychomotor behaviors, including increased exploratory locomotor activity and explosive jumping. Additionally, we have recently demonstrated that PACAP-KO exhibits defects in sensorimotor gating as measured by prepulse inhibition (PPI) of the startle response. It is remarkable that PACAP-KO displayed these marked behavioral abnormalities without marked changes in the basal levels of brain monoamines and their metabolites. In the present study, to address possible contributions of monoaminergic (dopaminergic and serotonergic) systems in these phenotypic abnormalities, behavioral responses to monoaminergic drugs (haloperidol, amphetamine, fluoxetine, 8-OH-DPAT) were examined in PACAP-KO. A potent dopamine (DA) D2-like receptor antagonist haloperidol (0.2 mg/kg) reduced the hyperactivity and jumping behavior, although the same dose of haloperidol did not reverse the deficit in PPI in PACAP-KO. In contrast, a psychostimulant amphetamine (2 mg/kg) effectively normalized

We have generated transgenic mice which express an enhanced green fluorescent protein (EGFP) under the control of the mouse glial fibrillary acidic protein (GFAP). In one of the transgenic lines, EGFP is observed exclusively in reactive astrocytes in sections of the neocortex after a cortical stab wound. Accordingly, it is suggested that these transgenic mice can be used as a tool to visualize reactive astrocytes. Using these animals, immunostaining of PACAP receptor (PAC1 receptor) was performed. PACAP is known to play important roles in neuroprotection, astroglial proliferation and differentiation. Therefore, the monitoring of the expression of PAC1 receptor after a cortical stab wound is necessary, because reactive astrocytes which are observed after brain injury are known to increase their number and to be involved in neuroprotection. As a result, we

Abstracts found dense immunostaining of PAC1 receptor in the reactive astrocytes at 5 days postsurgery, but not at 48 h. We further performed immunostaining of vimentin, the marker of immature glia, and gained the result that vimentin immunopositive cells were distributed in the area close to the needle track as PAC1 receptor immunopositive cells. These results suggest that PACAP is involved in the differentiation of reactive astrocytes induced in this brain injury model.

Phox2 proteins regulate VIP gene transcription Aviva Symes, Yasmin Bharucha and Xiuhaui Liu

57

decreased it in the evening. In males, there was a decrease of LH secretion due to PACAP treatment at night; however, PACAP did not influence LH secretion at the other time of the day. On the basis of our results, we concluded that PACAP might be involved in the circadian and episodic release of LH at pituitary level. This work was supported by OTKA grant T34429.

Expression of PAC1 receptor in rat thymus after irradiation N. Tokuda1, K. Hamasaki1, N. Mizutani1, Y. Adachi1, T. Sawada1, H. Funahashi2, S. Shioda2,3 and T. Fukumoto1

Department of Pharmacology, USUHS, Bethesda, MD, USA 1

Dept. of Human Science, Yamaguchi Univ. Sch. Med., Japan Dept of Anatomy, Showa Univ. Sch. Med., Japan 3 CREST of JST, Japan

VIP gene transcription is controlled by the combinatorial action of ubiquitous, cell-specific and inducible transcription factors that bind to specific sites on the VIP promoter. The gp130 cytokine family induces VIP transcription through the activation of AP-1 and Stat proteins that interact with a 180-bp cytokine response element (CyRE) in the VIP promoter. Interestingly, the anti-inflammatory cytokine transforming growth factor-h (TGF-h) also induces VIP gene transcription through the identical 180-bp CyRE, through the activation of distinct signaling pathways and transcription factors. TGF-h activates Smad transcription factors that translocate to the nucleus and bind the VIP CyRE. However, Smad binding to DNA usually requires accessory proteins that are gene-specific. We have identified the Phox2 homeodomain proteins as accessory proteins that bind together with Smad3 and Smad4 to the VIP CyRE to mediate VIP gene transcriptional induction by TGF-h. Mutation of either the Phox2 binding site or the Smad binding site abrogates the response of VIP CyREluciferase constructs to TGF-h stimulation suggesting that both homeodomain and Smad binding sites mediate the TGF-h effect. We have identified four other putative Phox2 binding sites within the VIP CyRE. Transfection of dominant negative Phox2 reduced the stimulation mediated by either gp130 cytokines or TGF-h through the VIP CyRE. Phox2a and Phox2b are neural specific homeodomain proteins that contribute to specifying sympathetic neuronal fate. As Phox2 proteins are expressed in parasympathetic and enteric neurons, where VIP is also expressed, Phox2a/2b may contribute to regulation of VIP gene transcription in a cell-specific manner, and in response to specific cytokines.

2

Effect of PACAP on LH release depends on the gender, on the time of day and in female rats on the day of the estrous cycle. Cell immunoblot assay study

High throughput screening of the genes regulated by PACAP during PC12 cell differentiation

´ . Nemeske´ri and K. Ko¨ves E. Szabo´, A

PACAP is known to show several actions that are important for the recovery of nerve injury. These days from many evidences it has been considered that nervous systems and immune systems are intimately related each other. Subsequently, PACAP may be also important for the regeneration from the injury of immune tissues. We irradiated rats sublethally and analyzed expression of PAC1-R (the G-protein coupled receptor specifically for PACAP) in the thymus after irradiation. We used female DA rats and irradiated with 8 Gy. After irradiation, thymus weight initially decreased and then increased rapidly. On days 14 to 21, thymus weights did not increase. After day 21, the thymus weights started to increase again and recovered to about 65% of control thymus weights. In the histological findings, the cell number decreased and the cortico-medullary junction was unclear after irradiation. The cell number and the structure recovered from day 7 and then seemed almost recovered on day 14. Immunohistochemically PAC1-R was detected on the cells of cortex and medulla in normal thymus. PAC1-R was expressed on epithelial cells and some types of round cells. On day 7 after irradiation, the expressions were strong. On day 14, the expression seemed almost same as normal control, but on day 21 it was strong again in the medulla. On day 28, the expression seemed to be almost recovered to the normal level. We also report the expression of changes of PAC1-R mRNA in the thymus after irradiation and discuss the possibility that PACAP may contribute to the regeneration of thymus after irradiation.

D. Vaudry1,2, A. Ravni1, Y. Chen2, C. Hamelink2, B.J. Gonzalez1, H. Vaudry1 and L.E. Eiden2 1

Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary

2

The presence of PACAP in the anterior pituitary of male and proestrous female rats and its partial colocalization with LH immunoreactivity were demonstrated. It was also shown by cell immunoblot assay (CIBA) that PACAP is released from the gonadotropes and in physiological concentration it could influence LH release from individual gonadotrop cells. In the present work, we studied whether the release of PACAP and the responsiveness of individual LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed and in female rats on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. The released hormone was trapped by the membrane and when immunostained they appeared in a form of a blot around the cells. We found that the number of PACAP releasing cells was higher in proestrous rats than in diestrous or in male rats and their number was always higher in the evening than in the morning. The effect of PACAP on LH cells was stimulatory in the morning of both proestrous and diestrous female rats (it enhanced the diameter of the blots). In proestrous rats, PACAP did not influence LH secretion in the afternoon or at night, but in diestrous rats it

PACAP promotes neurite outgrowth and inhibits proliferation of rat PC12 cells. Characterizing the PACAP differentiated PC12 cell transcriptome should provide genetic insight into how this process occurs in these cells, as well as in neuronal precursors. For this purpose, RNA samples were collected from PC12 cells before or after a 6-hr treatment with PACAP, from which labeled cDNA were hybridized to a highdensity array. The results indicate that the genomic response to PACAP involves at least 80 genes. Among the genes differentially expressed by PACAP, 71% were induced and 29% downregulated. Sixty-six percent of the messages affected by PACAP code for functionally categorized proteins, most of which were not previously known to be regulated during PC12 cell differentiation. PACAP has been shown to induce PC12 cell neurite outgrowth through the ERK pathway independently of the PKA. Therefore, treatments were conducted in the presence of the PKA inhibitor H89, or the MEK inhibitor U0126 in order to identify subsets of genes involved in PC12 cell differentiation. Co-treatment of PC12 cells with PACAP + H89 revealed a cluster of 5 genes specifically regulated

IFRMP 23, INSERM U413, Univ. Rouen, Rouen, France SMN, Lab. of Cell. and Mol. Reg., NIMH, Bethesda, MD, USA

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Abstracts

through the PKA pathway, and co-treatment of the cells with PACAP + U0126 revealed a cluster of 13 genes activated through the MEK pathway. Many of the known genes regulated by PACAP are known to be associated with neuritogenesis (i.e. annexin A2) or cell growth (i.e. GAS-1). Thus, some of the ESTs that exhibit the same regulation pattern may also be involved in the neuritogenic and antimitogenic effects of PACAP in PC12 cells.

Hypothalamic origin of centrifugal visual fibers in intact and pituitary stalk sectioned rats. Immunohistochemical and tracing studies V. Vereczki1, K. Fo´gel2, Zs. Boldogk} oi3, M. Kausz4, A. Lakatos4 and 4 K. Ko¨ves 1

Department of Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis University, Budapest, Hungary 2 Department of Surgery, St. John Hospital, Budapest, Hungary 3 Neuromorphological Laboratory, Hungarian Academy of Sciences, Budapest, Hungary 4 Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary The retinohypothalamic tract is well established; however, the presence of a reverse connection is still controversary. In the retina, centrifugal visual fibers were demonstrated but their origin is still not well known. Previously, we observed VIP fibers leaving the hypothalamus and running in the optic nerve. After removing the eyes, VIP immunoreactive cell bodies appeared in the magnocellular nuclei, which are not present in intact rats. We supposed that these cells are the origin of the VIP fibers in the optic nerves. In the present work we provided direct evidence for the cells of origin of this pathway using retrograde tracing techniques (cobaltic-lysin, biotynilated dextran, pseudorabies virus labeling) in intact and pituitary stalk sectioned rats. Co-ly was applied to the dissected end of the optic nerve, which remained connected to the hypothalamus. BDA and virus were injected into the eye. In the cases of Co-ly and BDA after a few days transportation time, in the case of virus 15 h later, we found retrogradelly labeled cells in the supraoptic and paraventricular nuclei supporting our previous assumption that those cells which send their axons to the eye are located in the magnocellular cell groups and these cells may give rise to the hypothalamoretinal pathway. This work was supported by OTKA grant T34429.

Differential transport of amyloid B peptides across blood brain barrier

Furthermore, AhP1 – 42 was effluxed via a saturable mechanism in young SAMP8 mice, while 1 – 40 did not show a saturable efflux. This shows that the transporter for AhP1 – 40 is altered in young SAMP8 mice. Aged SAMP8 showed total absence of efflux for AhP1 – 40 indicating that with aging the SAMP8 mice has an impaired efflux. The variations in the efflux of AhP among these three groups of mice supports the hypothesis that impaired transport could be a significant contributor to AhP accumulation within the brain and to the development of Alzheimer’s disease.

Specific upregulation of c-Maf and Jun B in Th2-polarized CD4 T cells of T cell-targeted VPAC2 receptor transgenic mice Julia Voice and Edward J. Goetzl Medicine-Microbiology, UC San Francisco, CA 94143, USA VIP regulates diverse functions of CD4 T cells, which express VPAC1 G protein-coupled VIP receptor constitutively, and VPAC2 after TCR stimulation. Transgenic C57Bl/6 mice (Tg), which constitutively express VPAC2 selectively in CD4 T cells exhibit an allergic phenotype as a result of a higher than normal Th2/Th1 cytokine ratio and a greater number of IL4 secreting T cells following TCR stimulation of CD4 T cells. The ability of VIP/VPAC2 to modulate transcriptional pathways involved in Th2 polarization was studied using CD4 splenic T cells from Tg and wild-type (WT) mice, stimulated in vitro by anti-CD3+ anti-CD28 antibodies. Following 48 and 96 hours of TCR stimulation, real-time PCR and western blot analyses showed a 2.5-fold mean increase in c-maf RNA and a 5.5-fold mean increase in nuclear c-Maf protein in Tg compared to WT mice. There was no difference in RNA or protein levels of GATA3, or the Th1 specific transcription factor, T-bet, between Tg and WT CD4 T cells. Jun B, a strong transcriptional activator of IL-4, which synergizes with c-Maf to activate the IL-4 promoter, was upregulated two-fold in Tg compared to WT CD4 T cells following 72-h stimulation. Incubation of Tg and WT CD4 T cells with catalytic VIPase IgG during T cell stimulation specifically prevented JunB and c-maf upregulation in Tg mice. Therefore, the interaction of endogenous T cell-derived VIP with VPAC2 enhances the Th2/Th1 ratio by specifically inducing the upregulation of the transcription factors Jun B and c-Maf and their related proteins. Supported by American Lung Association of California and NIH grant AI 29912.

Analysis of neuropeptide action on circadian rhythms using mice with targeted disruptions of the VIP/PHI and PACAP genes J.A. Waschek, S. Michel, J. Itri, V. Lelie`vre and C. Colwell

Sulekha Verma, Sandra Robinson, John E. Morley and William A. Banks Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, MO, USA Alzheimer’s disease is the most common cause of dementia in the elderly and its onset is difficult to diagnose. The physiopathology of the disease is complex and includes severe neuron and synapse loss, along with the accumulation of senile plaques, which contain the amyloid h peptides (AhP)1 – 40 and 1 – 42. There is evidence pointing to the AhP accumulation, likely due to impaired efflux from brain to blood, as a crucial event in the development of the disease. We have used the mouse model of aging (SAMP8), a mouse strain that accumulates AhP and develops cognitive impairment with age, to study the efflux of mouse AhP. AhP were radiolabeled with 131I by the chloramine-T method and purified by column chromatography. The purified monomeric peptides were injected into mice by the intracerebroventricular route. The rate of AhP efflux from the brain to blood was measured. We found that there was a reduction in the transport of AhP1 – 42 by both young and aged SAMP8 as compared to CD-1 mice.

Mental Retardation Research Center, Neuropsychiatric Institute, UCLA, Los Angeles, CA, USA PACAP and VIP appear to be essential for the regulation of circadian rhythms. PACAP is expressed in the retinohypothalamic tract and may (with glutamate) directly mediate the effects of light to the circadian center, the suprachiasmatic nuclei (SCN). VIP, on the other hand, is expressed within the SCN and may transmit light and other information to other neurons in the SCN and elsewhere in the brain. We have used physiological and gene disruption approaches to study the roles of these peptides in circadian function. Radioimmunoassay and immunohistochemistry confirmed a loss of peptide in the respective lines of mice. PACAP KO mice exhibit a normal circadian activity pattern in constant darkness, but have an altered phase shift in response to light. VIP KO mice have moderate to severe rhythm disturbances. Electrophysiological experiments indicate that PACAP may act to affect light shifts by facilitating AMPA-mediated neurotransmission, whereas VIP alters intracellular communication within the SCN by enhancing GABA neurotransmission.

Abstracts

Involvement of VPAC2 and PAC1 receptors in the cytosolic Ca2 + responses to subpicomolar PACAP38 in rat pancreatic B-cells H. Yamada, M. Watanabe and T. Yada Department of Physiology, Jichi Medical School, Japan It has been demonstrated that PACAP plays an important role in the regulation of insulin release in islets. Two types of PACAP receptors, PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R), are known to be present in pancreatic h-cells. PACAP stimulates insulin release and increases cytosolic Ca concentration ([Ca2 +]) in a concentration-dependent manner, in which peaks of the effects are observed at 10 13 and 10 9 M of the peptide. These findings suggested that the effect of PACAP at 10 13 M could be mediated by PAC1-R, while that at 10 9 M by VPAC2-R. This possibility was examined by studying the effects on [Ca2 +] in h-cells of PAC1-R-selective agonist and antagonist, maxadilan and M65, respectively, in comparison with those of PACAP38 and VIP. We isolated islets from Wistar rats and further dispersed into single hcells. [Ca2 +] in single h-cells was measured by fura-2 fluorescence imaging. In the presence of 8.3 mM glucose, PACAP38 increased [Ca2 +] in 111 out of 255 h-cells (44%) (mean amplitude: 126 nM). The incidence and amplitude of the [Ca2 +] response were slightly reduced to 42% and 115 nM, respectively, in the presence of M65. The results suggested that the major portion of the [Ca2 +] increase is independent of the PAC1-R. Maxadilan at 10 13 M increased [Ca2 +] in 14% of the cells. VIP at 10 13 M increased [Ca2 +] in 35% of the cells. Thus, when compared at the dose of 10 13 M, a VPAC-2 agonist VIP was more effective than a PAC1-R agonist maxadilan in inducing [Ca2 +] increases in h-cells. These results indicate that the [Ca2 +] responses to 10 13 M PACAP38 are mediated by both VPAC2-R and PAC1-R, in which the former appears to play a dominant role.

VIP: its role in myocardial fibrosis V.Z.C. Ye, G. Hodge, J.L.C. Yong and K.A. Duggan Hypertension Service and Dept. Anatomical Pathology, South Western Sydney Health Service, Sydney, NSW, Australia Concentrations of VIP in the heart are decreased in the later stages of both experimentally induced and human cardiomyopathy. However, it is not known whether the decrease in VIP causes the myocardial fibrosis or is a consequence of the fibrotic replacement of most of the myocardial tissue. To determine this we studied myocardial VIP concentrations in three animal models of early myocardial fibrosis, the WKY, the SHR and the L-NAME treated WKY. Each of the three groups (n = 18) was randomised to a low, intermediate or high salt diet (n = 6 each diet). Myocardial fibrosis increased

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with increasing dietary salt content (WKY: p < 0.001; SHR: p < 0.01; LNAME WKY: p < 0.005). In each of the models myocardial VIP concentration decreased (WKY: p < 0.01; SHR: p < 0.01; L-NAME WKY: p < 0.025) as myocardial fibrosis increased. That is myocardial VIP was inversely correlated with the degree of myocardial fibrosis. As we had previously demonstrated that inhibition of VIP metabolism by treatment with NEP and ACE inhibitors increased myocardial VIP concentrations we sought to determine whether increasing VIP in the heart by decreasing its metabolism was associated with a decrease in fibrosis. Myocardial VIP and fibrosis were determined in the L-NAME treated WKY fed a high salt diet and randomised to control or treatment with the NEP/ACE inhibitor omapatrilat. In the omapatrilat treated group myocardial VIP was increased ( p < 0.005) and myocardial fibrosis was decreased ( p < 0.005) compared with control. We conclude that decreased myocardial VIP is associated with increased myocardial fibrosis. Further, decreasing VIP metabolism and increasing VIP in the heart is associated with a decrease in myocardial fibrosis.

Long lasting smooth muscle relaxation by a novel PACAP analogue in human bronchi S. Yoshihara1, Y. Yamada1, T. Abe1, K. Kashimoto2, A. Linde´n3 and O. Arisaka1 1

Dept. of Pediatrics, Dokkyo Univ. School Med., Japan Pharm. Group, Health Sci. Div. of ITOHAM, Japan 3 Lung Pharm. Group, Dept. of Resp. Med. and Allergol., Go¨teberg Univ., Japan 2

We compared the relaxant effect of pituitary adenylate cyclase activating peptide (PACAP)1 – 27 with that of a newly developed PACAP1 – 27 analogue, [Arg152021Leu17]-PACAP-Gly-Lys-Arg-NH2, in human bronchi in vitro. In human bronchi precontracted by carbachol (0.1 AM), cumulative administration of PACAP1 – 27 and salbutamol caused concentration-dependent smooth muscle relaxation with similar potencies and maximum relaxant effects. Non-cumulative administration of the PACAP1 – 27 analogue and the original PACAP1 – 27 caused concentration-dependent relaxation with a similar maximum relaxant effect and potency as well. However, the onset and offset of action was markedly slower for the PACAP1 – 27 analogue than for the original PACAP1 – 27 (>90% vs. < 10% of peak relaxation remaining 5 h after administration). Peptidase inhibition by captopril (10 AM) and phosphoramidon (1 AM) significantly increased the maximum relaxant effect and duration of action of PACAP1 – 27 but not of the PACAP 1 – 27 analogue, during the 3 h of observation in the human bronchi. We conclude that [Arg152021Leu17]-PACAP-Gly-Lys-Arg-NH2 produce significant, concentration-dependent and sustained airway smooth muscle relaxation in vitro. The sustained relaxant effect is due, at least in part, to the PACAP1 – 27 analogue being less susceptible to cleavage by peptidases than the original peptide PACAP1 – 27.

Dr. Noboru Yanaira Memorial Session

Abstracts

Researches on salivary secretion of chromogranin A by the late Professor Noboru Yanaihara at Yanaihara Institute T. Kanno Professor emeritus of Hokkaido University, Hokkaido, Japan Professor Yanaihara et al. developed region-specific immunoassay for rat and human chromogranin A (CgA), and revealed that CgA-like immunoreactivity (IR) is a sensitive biochemical index of psychological stress response. We have then confirmed that the source of salivary CgAIR is in the granular duct cells of rat salivary gland, and is vigorously secreted into the saliva during activation of the efferent sympathetic/ adrenomedullary system in the stress response. The CgA-IR secretion coincides with vigorous compound exocytosis of secretory granules and redistribution of subcellular IP3-receptor type 2 in the granular duct cells. VIP (0.1 – 1.0 nM) in the presence of 0.1 AM ACh causes gradual CgAlike IR secretion. The mode of secretion resembles that induced by isopreterenol, a h-adrenergic agonist, suggesting cAMP is commonly be involved.

Physiological roles of PACAP in the central nervous system and endocrine disruptors Yoshinori Masuo1,2, Syuichi Oka1, Masatoshi Morita3 and Masami Ishido3 1 National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan 2 NEDO, Tokyo, Japan 3 National Institute for Environmental Studies, Tsukuba, Japan

In 1990s, we investigated physiological profiles of PACAP in the central nervous system. Regional distribution of PACAP and its binding sites in the adult rat brain and ontogeny of these parameters suggested that the peptide might play physiological roles as a neurotransmitter/modulator. The highest level of PACAP content was found in the suprachiasmatic nucleus with the day – night variation. With dialysis technique, we found that PACAP and VIP stimulated the spontaneous release of acetylcholine from the hippocampus. An i.c.v. injection of PACAP caused behavioral hyperactivity and counteracted reserpine-induced hypothermia, which was more potent than VIP and TRH. It was reported that PACAP stimulates dopamine release from PC12 cells. These data suggest that PACAP may have roles as an endogenous psychostimulant. It is known that psychostimulants inhibit behavioral hyperactivity seen in patients with pervasive developmental disorders, including autism, and attention-deficit hyperactivity disorder. Recently, we are studying the effects of endocrine disruptors in collaboration with Yanaihara Institute. An intracisternal injection of endocrine disruptors into neonatal rats abolished the development of dopaminergic neurons, which resulted in hyperactivity. The effects of these compounds are now examined on dopamine release from PC12 cells. On the other hand, it was demonstrated that PACAP-knockout mice showed behavioral hyperactivity. Taken together, PACAP may be involved in mechanisms of hyperkinesia caused by the deficit in the development of dopaminergic neurons.

Tribute to Professor Noboru Yanaihara W. Roste`ne and G. Rosselin INSERM U339/INSERM U55, Hoˆpital St. Antoine, 75012 Paris, France On October 23, 2001, disappeared with N. Yanaihara a part of the most wonderful odyssey in biomedical science, the discovery and

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implication of peptides in peripheral and brain functions. N. Yanaihara was not only considered as a leader in this field of peptide research for the discovery of these peptides, synthesis, understanding of their processing, and development of invaluable antibodies, but also as a source for many scientists around the world of useful tools, which led to an increase in knowledge and impressive discoveries. The quality of his work is attested by 486 publications referred in PubMed and several books, which mark a milestone in history of peptides. His charming and laughing personality attracted several investigators from Japan and other places in the world to directly work with him. He was also a great entrepreneur and built in a heavenly place a laboratory that became the Yanaihara Institute. All his friends, collaborators and young researchers, which still profit of the concepts and technologies developed by N. Yanaihara, will be present in this symposium to honor one of the most famous Japanese scientists.

Remembering Professor Yanaihara Sami I. Said State University of New York at Stony Brook and VA Medical Center, Northport, NY, USA For me, the pleasure of returning to Japan after a long absence is diminished by the knowledge that Noboru Yanaihara will not be there. Like his many other friends and colleagues around the world, I will miss his smiling face, his warm personality and his great energy. It was Noboru Yanaihara who, for some 20 years, planned, organized and hosted many excellent symposia on gastrointestinal and neuropeptides. Scientists from everywhere came together to discuss their research. At the same time, they also enjoyed a taste of Japanese culture and traditions, and appreciated the kind hospitality and organizational skills of Noboru and his wife and close associate, Professor Chizuko Yanaihara. Noboru’s numerous and important contributions to peptide chemistry and biology will be long remembered through his published works, and through his students and trainees. We are saddened that he left us prematurely, but his spirit lives in his wife, his son Dr. Hitoshi Yanaihara and the rest of the Yanaihara family.

VIP and NO in the pancreas Tooru Shimosegawa MD Division of Gastroenterology, Tohoku University Graduate School of Medicine, Japan To memory of the late Professor Noboru Yanaihara, I express my great gratitute to his courtesy and encouragement. The late Professors Noboru Yanaihara and Shigeru Kobayashi had opened out for me the way to research and provided also a chance to study under Professor Sami I. Said. Since my specialty is the pancreas, I was attracted by the delicate intrinsic nervous system in the pancreas. By immunohistochemistry, VIP and NADPH-diaphorase activity, a marker of NO synthase, were colocalised in a portion of neurons in the pancreas, though their functional distinction in pathophysiology being still obscure. In models of acute pancreatitis, NO is overproduced by inflammatory cells through the activation of NF-kB, and in vitro studies suggested inhibitory roles of NO on the expression of various proinflammatory molecules. Studies on pancreatic stellate cells, a key for the mechanism of fibrosis, suggested a participation of various factors including them on the regulation.

Satellite Symposium on GPCRs Hakone, Japan September 4, 2003

Abstracts

Pituicytes in the posterior pituitary gland are new member of peptide hormone secreting cells

Localization of metastin and its receptor-like immunoreacivity in the rat spinal cord

S. Adachi1, K. Fujiwara1,2 and K. Inoue1

S. Endo1, T. Nakamachi1, L. Yin1,3, H. Ohtaki1,3, T. Ohtaki2 and S. Shioda1,3

1 2

Dept. of Regulation Biol., Fac. of Sci., Saitama Univ., Japan Dept. of Physiol., Jichi Med. Sch., Japan

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1

Department of Anatomy, Showa University School of Medicine, Japan Pharmaceutical Discovery Research Division, Takeda Chemical Industries Ltd., Japan 3 CREST of JST, Japan 2

Pituicytes are known to surround terminal processes of neurosecretory cells and to act as supportive cells in the posterior pituitary gland (PP). Pituicytes contain many lipid droplets in their cytoplasm. It also known that pituicytes response to dehydration and change their morphological appearances. Recently a novel peptide comprising 60 amino acid residues was isolated from the porcine hypothalamus as a ligand of galanin receptor subtype, GalR2. This peptide is called galanin-like peptide (GALP). To study of the localization of GALP, we performed immunocytochemistry and found that pituicytes in the PP produce GALP. In this poster, we report that pituicytes in the PP are new member of peptide hormone producing cells. Adult Wistar male rats were deeply anesthetized and perfused with 4% paraformaldehyde. To examine the localization of GALP in the pituitary gland, we prepared a normal group and a water deprivation (4 days) group. Some the rats in these groups were injected with colchicine (i.p.) 1 day before sampling. Frozen pituitary sections were immunocytochemically stained with specific monoclonal antibodies for GALP. In turn, frozen pituitary sections were subject to double immunostaining of GALP and S-100 protein. Furthermore, we observed GALP-ir cells in the PP on electron microscopy. GALP-ir cells were localized in the PP, but not in the anterior or intermediate pituitary lobe. We observed the response to dehydration and found that GALP-ir cells in the PP dramatically increased in the PP of dehydrated rats. On double immunostaining of S-100 protein and electron microscopic observation, we found that GALP was produced in pituicytes.

Molecular characterization of a novel variant of type I PACAP receptor which is not coupled to adenylyl cyclase 1

1,2

1

1

2

Y. Anouar , J. Chu , D. Alexandre , B.J. Gonzalez , B.K.C. Chow and H. Vaudry1 1 Eur. Inst. Pept. Res. (IFRMP 23), Lab. Cell. Mol. Neuroendocrinol., INSERM U413, Univ. Rouen, Mont-St-Aignan, France 2 Dept. of Zoology, Univ. Hong Kong, Hong Kong, China

PACAP acts through a type I receptor, PAC1-R, specific for PACAP, and two type II receptors, VPAC1-R and VPAC2-R, that bind PACAP and VIP with high affinity. Several PAC1-R splice variants have been described that presumably fine-tune the effects of PACAP in certain tissues. We have recently identified a novel PAC1-R splice variant in the frog Rana ridibunda. This receptor isoform, named PAC1-Rmc, displays a C-terminal tail whose sequence is distinct from that of the PAC1-R C-terminus due to the insertion of a cassette of 13 nucleotides downstream of the sequence encoding the seventh transmembrane domain. PAC1-Rmc is highly expressed in the central nervous system of the frog. However, PACAP failed to activate adenylyl cyclase in cells transfected with PAC1-Rmc. Fusion of PAC1-Rmc with GFP revealed that this receptor, as PAC1-R, is targeted to the plasma membrane. We have recently generated two stable CHO cell lines that express either PAC1-R or PAC1-Rmc. We confirmed in these cell lines the effect of PACAP on adenylyl cyclase through PAC-1R and the lack of effect of the peptide in cells expressing PAC1-Rmc. In addition, we showed that the two receptor variants are able to bind iodinated PACAP with high affinity. Experiments are in progress to determine whether PACAP is able to activate other signaling mechanisms via PAC1-Rmc and whether this variant may act as a negative dominant of PAC1-R, at least in regard to activation of adenylyl cyclase. Supported by INSERM (U413) and a France – Hong Kong exchange program (Procore).

Metastin is a novel peptide that is recently isolated from human placenta as the endogenous ligand of an orphan G-protein coupled receptor, hOT7T175 (AXOR12 and GPR54). Metastin is encorded by putative metastasis suppressor gene named KiSS-1 and consists of 54amino acid residues and an amidated C-terminus. Metastin is shown to inhibit chemotaxis, cell motility/growth, and invation of CHO cells transfected with hOT7T175 cDNA in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. However, the localization of metastin and its receptor (hOT7T175) in the central nervous systems is poorly understood. Therefore, we studied distribution and localization of metastin and its receptor in the rat spinal cord to assume the possible functions of metastin in the spinal cord. The frozen sections of the rat spinal cord were immunostained by using free-floating methods. In the coronal sections of the lumbar level, the immunoreactivity of metastin was noted. The small immunopositive cells with processes were scattered in the white matter. On the other hand, the immunoreactivity of metastin receptor was noted similarly. Scattered immunopositive somata were distributed widely in the white matter, conspicuously in the funiculi anterior or lateralis. The motoneurons in the ventral horn were also immunoreactive for its receptor. We are now studying what kind of cells express metastin and its receptor by using a double immunostaining method.

Study on the prolactin-releasing peptide (PrRP) producing cells in the area postrema and adrenal gland K. Fujiwara1,3, H. Matsumoto2, T. Yada3 and K. Inoue1 1

Dept. of Regulation Biol., Saitama Univ., Japan Discovery Research Lab. I, Pharmaceutical Research Division, Takeda Chem. Industry, Ltd., Japan 3 Dept. of Physiol., Jichi Med. Sch., Japan 2

Prolactin-releasing peptide (PrRP) is a novel hypothalamic peptide that stimulates prolactin release from anterior pituitary gland. PrRP is also known to play a role as an important neurotransmitter and/or neuromodulator in the central nervous system (CNS). In line with this, we have recently found that PrRP acts as an important stress mediator in the CNS. In the normal adult rat brain, PrRP neurons are known to be located only in three areas: the dorsomedial hypothalamic nucleus, the ventrolateral reticular formation and the nucleus of the solitary tract in the medulla oblongata. These PrRP neurons project neurites into the various brain areas. The area postrema (AP) is known as a site where PrRP neurons project. In this area, PrRP receptor mRNA is expressed at a high level, which suggests that PrRP may play a role in this area. In this study, by using in situ hybridization and immunocytochemistry, we have demonstrated that PrRP-producing cells appear in the AP of the rats after adrenalectomy. In addition to this, we have detected PrRP mRNA in the rat adrenal medulla by PCR. Subsequently, we sought to identify PrRP-producing cells in the adrenal medulla by immunocytochemistry. Our results revealed that the PrRP immunopositive cells were indeed immunoreactive for phenylethanolamine N-methyltransferase, which demonstrates that the PrRP-producing cells in the adrenal gland are adrenalineproducing cells. These results therefore indicate that PrRP may play a role in relating the adrenal gland to the area postrema.

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Abstracts

Morphological analysis of ghrelin and its receptor in the rat pancreas H. Funahashi1, S. Kato1, J. Sakurai2 S. Inoue2, S. Nakajo3, Y. Date4,5, M. Nakazato4, K. Kangawa5 and S. Shioda1 1

Dept. of Anatomy, Showa Univ. Sch. Med., Japan Dept. of Physiol. and Nutrit., Kyoritsu Women’s Univ., Japan 3 Lab. of Biol. Chem., Showa Univ. Pharm. Sci., Japan 4 Third Dept. of Internal Med., Miyazaki Med. College, Japan 5 Dept. of Biochem., National Cardiovascular Center Res. Inst., Japan 2

Ghrelin is a novel peptide, which was first purified from the rat and human stomach, and has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Ghrelin is considered to have very important roles in feeding regulation as well as GH release. Northern blot analysis of rat and human tissues reveals the highest level of ghrelin and GHS-R mRNA in the stomach, followed by the pancreas, duodenum, jejunum, lung, liver and brain. Most recent physiological experiments in the pancreas have shown that ghrelin regulates insulin secretion. However, action cites of ghrelin in the pancreas are not yet identified. In this study, to gain insight into the role of ghrelin in the rat pancreas, we examined the localization of ghrelin and GHS-R with immunohistochemistry. Both ghrelin and GHS-R immunoreactivities were detected mostly in the same cells in the peripheral region of the islets of Langerhans. Many and they were overlapped with glucagon immunoreactivities. Moreover the weak GHS-R immunoreactivity was found in insulincontaining B cells. These observations suggest that ghrelin is synthesized and secreted from A cells to regulate insulin secretion from B cells. In addition, ghrelin may act on A cells to function as an autocrine manner.

Sphingosine 1-phosphate (S1P) and the S1P1(Edg-1) G protein-coupled receptor (GPCR) constitute a unique immunoregulatory system Edward J. Goetzl and Markus H. Graeler University of California, San Francisco, CA 94143-0711, USA Human and mouse CD4 T cells in thymus, spleen and blood preferentially express high levels of S1P1 and S1P4 GPCRs, which are suppressed by activation of the T cells through stimulation of the T cell antigen receptor (TCR). S1P effects on naı¨ve and memory CD4 T cells include elicitation of direct chemotaxis (1 – 100 nM), inhibition of secretion of IFN-gamma and IL-4 but not IL-2 (0.1 – 1 uM), reduction in proliferation (1 nM – 1 AM), and suppression of chemotactic responses to diverse chemokines (0.3 – 3 AM). Results of genetic and pharmacological studies demonstrate that S1P1, but not S1P4, receptors are the predominant transducers of S1P effects on T cells. T cell-targeted S1P1 GPCR transgenic mice show numerous aberrations in blood and tissue levels of T cells and in T cell-dependent immune responses. That the principal T cell effects of the S1P-S1P1 receptor axis are directed to migration and tissue localization suggest the possibility of novel approaches to therapy for autoimmune diseases and transplant rejection.

Alterations by endocrine disruptors of gene expression of G-protein-coupled receptors in the rat brain Masami Ishido1, Masatoshi Morita1, Syuichi Oka2 and Yoshinori Masuo2,3

Gene manipulation studies have developed many hyperactive animal models. It was shown in various knockout mice lacking the gene for a calcium calmodulin kinase II, receptors of serotonin 1A, 1B, dopamine D4, M1 muscarinic acetylcholine, PACAP, a brain-derived neurotrophic factor, or Go protein. These facts allowed us to examine if G protein-coupled receptors (GPCR) might be involved in the chemical-caused hyperactivity. To this end, we performed DNA array analyses, using BD Atlas Rat 1.2 DNA array membranes (BD Biosciences Clontech) that have 120 cDNAs of GPCR, including orphan receptors. It was found that some endocrine disruptors altered the gene expression of GPCR. Dibutylphthalate (DBP) changed the gene expression of dopamine D1A, D2 and D4 receptors. It was particularly notable that gene expression of galanin receptor 2 was largely decreased by nonylphenol, DBP or diethylhexylphthalate (DEHP) with 3.6-, 8.7- and 3.8-fold, respectively. Bisphenol A slightly (1.5-fold) increased the levels of gene expression of serotonin 1B receptor. Galanin acts as a neuromodulator in the neural circuitry, mediating cognition, learning and memory and reduces the evoked release of neurotransmitters such as dopamine. It is therefore tempting to examine if the endocrine disruptors might exert on such activities of galanin or if the neuropeptide is further involved in hyperactivity in the rat.

Neuronal mechanism of feeding regulation by galanin-like peptide H. Kageyama1, F. Takenoya1,2, H. Funahashi1, K. Inoue3, T. Ohtaki4, S. Inoue5 and S. Shioda1 1

Dept. of Anatomy, Showa Univ. Sch. Med., Japan Dept. of Physical Educ., Hoshi Univ. Sch. Pharm. Pharm. Sci., Japan 3 Dept. of Reg. Biol., Fac. of Sci., Saitama Univ., Japan 4 Pharm. Res. Div., Takeda Chem. Ind. Ltd., Japan 5 Dept. of Nutr. Physiol., Fac. of Home Econ., Kyoritsu Women’s Univ., Japan 2

Newly discovered Galanin-like peptide (GALP) shares sequence homology with galanin. GALP is one of the neuropeptides that are implicated in neuroendocrine regulation of body weight and food intake. GALP in the brain is expressed in neurons in the arcuate nucleus (ARC) and nerve terminals in the median eminence. Intracerebroventricular (i.c.v.) injection of leptin is shown to increase the number of GALP mRNA-expressing neurons in the ARC, suggesting that GALP may suppress food intake. However, injection of GALP into the paraventricular nucleus (PVN) increases the food intake. The function of GALP is not yet determined. The purpose of this study is to determine which area and what neurons are activated by GALP. In this study, male Wistar rats were used. GALP (3 nmol) or vehicle was injected into the lateral ventricle. The animals were fixed by fixative containing 4% paraformaldehyde 1 h after injection. Fresh frozen sections were prepared and then stained by a double fluorescence immunohistochemistry using antibodies against c-Fos, tyrosine hydroxylase (TH), orexin and melanin-concentrating hormone (MCH). GALP induced Fos-positive cells in periventricular zone of the third ventricle. Some TH-positive neurons showed Fos-immunoreactivity in the ARC. However, orexin- or MCH-positive neurons did not show any Fos-immunoreactivity. In conclusion, GALP may stimulate food intake via TH-containing neurons in the hypothalamus.

Visualization of an activity-dependent local secretion of BDNF

1

Natl. Inst. for Environ. Studies, Tsukuba 305-8506, Japan Natl. Inst. of Advanced Industrial Sci. and Technol., Tsukuba 305-8566, Japan 3 NEDO, Tokyo, Japan 2

Recently, we demonstrated that hyperactivity was induced by intracisternal administrations of some endocrine disruptors into the neonatal rat.

M. Kojima AIST, Japan We have developed a plasmid DNA encoding GFP-labeled BDNF and expressed this fusion protein (BDNF-GFP) in cultured neurons, slice

Abstracts preparations and intact brains (Kojima et al., 2002). One of the unsolved questions concerning neurotrophin secretion is whether the activity-dependent secretion of neurotrophins can occur locally. If this is the case, neurotrophins could strengthen the connectivity between specific neurons. To study this possibility, we examined the effect of local stimulation with caged-glutamate on the culture of BDNF-GFP expressing hippocampal neurons. To examine the activity-dependent local secretion of BDNF, we photolysed caged glutamate in a defined area of dendrites (proximal) of BDNF-GFP expressing neurons, and then compared the intensity with that of distal dendrites without stimulation (distal). In the presence of TTX, the fluorescence of BDNF-GFP clusters was found to disappear in the proximal sites with local stimulation, whereas no significant change in BDNF-GFP intensity was observed in the distal sites. UV-flash to this opposite site later confirmed its ability of BDNF-GFP secretion. This result likely provides a suggestion of activity-dependent local secretion of BDNF, as well as a hypothesis of input-specific regulation of BDNF secretion.

Physiological functions of two appetite regulating peptides; ghrelin and neuromedin U Masayasu Kojima Molecular Genetics, Institute of Life Science, Kurume University, Japan Using GHS-R expressing cell line, we purified a peptide from stomach that potently activated GHS-R. The purified peptide was consisted of 28 amino acids, in which the Ser-3 residue was n-octanoylated. Surprisingly, this n-octanoyl modification at Ser-3 was essential to the activity, since desacyl-peptide could not induce [Ca2 +]i changes. We named the GH-releasing peptide ‘‘ghrelin’’ (‘‘ghre’’ is the Proto-Indo-European root of the word ‘‘grow’’). In rat brain, ghrelin immunoreactive neurons were found to be localized in the hypothalamic arcuate nucleus, a region where regulates food intake. In fact, i.c.v. injection of ghrelin strongly stimulated feeding in rats and increased body weight gain. These facts suggest that ghrelin is a new physiological regulator of nutritional homeostasis. GPR66 is an orphan G-protein coupled receptor (GPCR), whose structure is similar to the ghrelin and motilin receptors. We have tried to purify a natural ligand for GPR66 in rat tissues and identified a 23-amino acid peptide as the endogenous ligand. Sequence analysis revealed the peptide as neuromedin U (NMU), a smooth-muscle-contracting peptide that was first purified from porcine spinal cord by our group. NMU binds to GPR66-expressing cells with high specificity to induce intracellular calcium mobilization. When NMU was injected intracerebroventricularly (i.c.v.) into rats, it potently suppressed food intake. In contrast, ICV injection of NMU-antibody increased food intake. These results suggest that NMU is a potent endogenous anorexic peptide.

GPCR catalogue: preparation of cDNA or protein library which is enriched in and represents GPCRs T. Kubo, M. Takeda and S. Kobayashi Molecular Neurophysiology, Neuroscience Research Inst., National Inst. Advanced Industrial Sci. and Tech. (AIST), Japan G protein coupled receptors (GPCRs) mediate essential processes in cell signaling, reception and transduction of a specific signal from outside and initiation of intracellular reactions. As the genome projects are reaching completion, we will soon get a complete list of GPCRs. The GPCR catalog with molecular entities, however, is an acute demand for drug screening, and for studying cell-signal networks. We have developed a new method to construct a cDNA library, in which a family of functional proteins such as GPCRs, ion channels, transporters,

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proteinases, proteinase inhibitors, growth factors, etc. are highly enriched (Patent No. 3243531). Applying this technique, we constructed a GPCR cDNA library. The library was evaluated by sequencing 36 colonies at random. They included a series of 5-HT receptor subtypes and two orphan receptors. The cDNA library is transcribed and translated to express a family of functional GPCRs, thus suitable for activity-based screening of a target protein for a specific drug, chemical substance, taste or odorant molecule, a peptide ligand and even an interacting protein on the GPCR-mediated signaling pathway.

Galanin-like peptide (GALP) increases cytosolic Ca2 + in neurons immunoreactive for growth-hormone releasing hormone (GHRH) in the arcuate nucleus M. Kuramochi1,2, D. Kohno1, H. Kato2 and T. Yada1 1 2

Dept. of Physiol., Jichi Med. Sch., Japan Dept. of Psychi., Jichi Med. Sch., Japan

Galanin-like peptide (GALP), discovered in the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC). GALP stimulates luteinizing hormone (LH)-releasing hormone-mediated LH secretion. However, whether GALP could stimulate secretion of another peptides in the hypothalamus are not known. Intracerebroventricular injection of GALP was shown to stimulate food intake in the rats. However, the mechanism underlying the orexigenic effect of GALP is unknown. We investigated the effects of GALP on cytosolic free Ca2 + concentration ([Ca2 +]i) in the neurons isolated from the ARC, a feeding-controlling center. GALP at the concentration of 10 10 M increased [Ca2 +]i in some (14%) of the ARC neurons. Some of these GALP-responsive neurons also responded to ghrelin. To determine what cell types responded to GALP, [Ca2 +]i measurements were followed by immunocytochemical staining with antiserum for neuropeptide Y (NPY), proopiomelanocortin (POMC), growth hormone-releasing hormone (GHRH), the peptides that are localized in the ARC. Some of GALP- and ghrelin-responsive cells contained GHRH. In contrast, GALP-responsive neurons overlapped neither with NPY- nor with POMC-containing neurons. These results indicate that GALP may regulate GHRH neurons in the ARC and suggest that the orexigenic effect of GALP is not mediated by NPY and POMC neurons.

Links between reproduction and feeding: a concept derived from the rat model Kei-ichiro Maeda and Hiroko Tsukamura Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601 The energy level is one of the critical factors regulating feeding behavior as well as reproductive functions, such as follicular development and ovulation. The present paper deals with the role of metabolic fuels as signals regulating feeding and reproduction reciprocally. Pharmacological blockade of the glucose or fatty acid metabolism induces feeding behavior and suppresses reproductive functions, suggesting that oxydizable metabolic fuels, such as glucose and free-fatty acids are the metabolic signals conveying the energy information. Our previous studies suggest that the energy availability could be detected by a sensor localized in the hindbrain to control both the feeding and reproduction during energydeprived condition. The noradrenergic pathway could play a key role in transmitting the information to the hypothalamic paraventricular nucleus from glucose-sensing cells in the hindbrain. Glucokinase and glucose transporter 2, which consist of a glucosesensing unit in the pancreatic B cell, have been found in ependymocytes of

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Abstracts

the cerebroventricular wall of the hindbrain. Thus, the ependymocyte may serve as a energy sensor and convey the energy information to noradrenergic neurons to regulate feeding and reproduction. Recent results on the effect of some orexigenic peptides on the reproductive functions will be also discussed.

septum and substantia innominata as well as in dopaminergic neurons in the substantia nigra as well as in hypothalamic nuclei involved in vasopressin regulation. These original data suggest that chemokines may represent a new class of neuromodulatory peptides in CNS and neuroendocrine functions.

Orexin receptor expression after brain ischemia

G protein-coupled receptors having DY-sequence in the amino-terminal region act as coreceptors for human and simian immunodeficiency viruses: identification of the functional domain of the coreceptor activity of GPR1

T. Nakamachi1,2, H. Ohtaki1,2, K. Dohi1,2, L. Yin1,2, E. Sakura1, F. Kudo1, H. Funahashi1 and S. Shioda1,2 1 Department of Anatomy, Showa University School of Medicine, Tokyo, Japan 2 The Core Research for Evolution Science and Technology (CREST) of Japan Science and Technology (JST), Japan

Introduction: Orexin is a neuropeptide that regulates feeding behavior, sleep-awaking cycle and other unknown physiological functions. Recently, it is reported that orexin receptor-1 (OX1R) level is increased after focal ischemia in rat. However, it has not been precisely demonstrated morphological changes of OX1-R immunoreactivity after brain ischemia. Therefore, the purpose of this study is to study the morphological changes of OX1-R immunoreactivity in mice brain after forebrain ischemia. Method: C57BL/6 mice were occluded bilateral common carotid artery for 25 min and reperfused. The animals were sacrificed at 0, 4, 8, 16, 24, 48, 96 or 168 h following ischemia/reperfusion. The brains from these animals were removed and immunostained with OX1R antibody. Morphological evaluation of the apoptotic cells was studied with toluidine blue staining and TUNEL labeling. Results and discussion: A part of pyramidal cells of hippocampal CA1 region began to fall out 96 h after ischemia and the CA1 pyramidal cells had fallen out broadly 168 h after ischemia. Moreover, the TUNEL-positive cells were observed in the CA1 area 96, 168 h after ischemia. Although the immunoreactivity of OX1R was decreased 4 – 24 h after ischemia, it was increased predominantly and peaked at 48 h after ischemia in the hippocampus and neocortex. These results suggest that orexin may play very important roles in delayed neuronal cell death.

Brain localization of chemokines and GPCR chemokine receptors: possible implication in neuroendocrine communication and neuro-degenerative diseases W. Roste`ne, G. Banisadr, D. Skrzydelski, R.M.Gosselin, P. Kitabgi, S. Me´lik Parsadaniantz.

N. Shimizu, A. Tanaka, A. Oue, K. Kanbe and H. Hoshino Dept. of Virology and Preventive Medicine, Gunma Univ. Sch. Med. Human immunodeficiency virus type-1 (HIV-1), HIV type-2 (HIV-2) and simian immunodeficiency virus (SIV) infect target cells by using CD4 as the main receptor and several G protein-coupled receptors (GPCRs) as coreceptors. More than 10 GPCRs including chemokine receptors have been reported to work as coreceptors of HIV-1, HIV-2, or SIV strains. Chemokine receptors, CXCR4 and CCR5, act as coreceptors for T-cell line(T)-tropic (X4), and macrophage(M)-tropic (R5) HIV-1, respectively. We isolated an HIV-1 variant, GUN-1V, from a T- and M-tropic (dualtropic) (R5-X4) HIV-1 strain, GUN-1WT. GUN-1V infected primary brain-derived CD4-positive pericytes (BT-cells) that are resistant to infection with M-, T- or dual-tropic (R5, X4 or R5 – X4) HIV-1 strains. Most of the coreceptors contain tyrosine-aspartic acid (DY) sequences in their amino(N)-terminal regions. In GPCRs having this characteristic, an orphan GPCR, GPR1, was identified as a coreceptor which determines the BT-cell tropism of GUN-1V, because when a CD4-positive human glioma-derived cell line, NP-2/CD4, which is resistant to infection with diverse HIV-1, HIV-2, and SIV strains, had been transduced with GPR1, the cell line became susceptible to GUN-1V but not to GUN-1WT. Several mutants of the N-terminal region of GPR1, such as amino acid substitutions, deletions, and chimeras with CCR5, were constructed to identify functional domains for the coreceptor activity. Small domains containing four tyrosine residues in the N-terminal region are important for the coreceptor function of GPR1.

The role of nicotine and prolactin-releasing peptide (PrRP) on stress mediation in the central nervous system Binggui Sun1, Sachika Adachi1, Haruki Nemoto1, Ken Fujiwara2 and Kinji Inoue1

INSERM E 0350, Hoˆpital St. Antoine 75012 Paris, France 1

Chemokines are proteins that chemoattract and activate immune and non-immune cells both in vivo and in vitro. Few recent reports showed that they may also play a role in the brain (CNS). The understanding of their role in brain functions needs a detailed anatomical distribution, which is still missing. We thus determine the neuroanatomical and cellular localization of the two main families of chemokines (CC and CXC), on the one hand, MCP-1/CCL2 and its receptor CCR2, and on the other hand, SDF1/CXCL12 and its receptor CXCR4 in the adult rat brain by immunohistochemistry. We show that SDF-1 and MCP-1 as well as their respective GPCR receptors CXCR4 and CCR2 are constitutively expressed in the rat CNS. In contrast to what was thought, they are found not only in glial cells but also in neurons in discrete brain regions. Indeed, SDF-1 is mainly found in cortex, globus pallidus, substantia innominata, hippocampus, hypothalamus, substantia nigra and occulomotor neurons, whereas MCP-1 is expressed in the substantia nigra, hypothalamic and occulomotor neurons. Interestingly, CXCR4 is found in the same regions as SDF-1, whereas CCR2 presents a distribution slightly different from that of MCP-1. SDF-1 and CXCR4 as well as MCP-1 and CCR2 are highly expressed in cholinergic neurons of the

Department of Regulation Biology, Faculty of Science, Saitama Unviersity, Japan 2 Department of Physiology, Jichi Medical School, Japan The prolactin-releasing peptide (PrRP) was recently found to be an orphan receptor ligand which stimulates prolactin release in the anterior pituitary gland. However, projections of the PrRP neurons are not found in the external region of the median eminence, a location where hypophysiotropic hormones are known to be secreted. But it is known that PrRP is produced in the noradrenergic A1 and A2 neurons in the medulla oblongata. It is also known that stress activates PrRP positive neurons in the A1 and A2 regions and thereby stimulates ACTH secretion through CRH neurons that in turn activate the hypothalamo – pituitary – adrenal axis. In the present study we found that restraint stress after nicotine injection can dramatically activate neurons in the A2 region and increase cFos immunoreactive cells in both PrRP and non-PrRP neurons. However, restraint stress after saline injection only stimulates the PrRP immunoreactive neurons in the A2 region. Furthermore, the injection of nicotine without stress produced less effect on the neurons in the A2 region than that of nicotine injection followed by stress, and stress without nicotine

Abstracts injection stimulates only the PrRP positive neurons. These results suggest that nicotine can increase the sensitivity to the stress response in the A2 region of the medulla oblongata.

Overview of GPCRS, comprehensively predicted from human genome

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GALP-containing neuron network system in the rat hypothalamus F. Takenoya1,2, H. Kageyama1, H. Funahashi1, H. Matsumoto3, T. Ohtaki3, S. Katoh1, M. Takeuchi2 and S. Shioda1 1

Dept. of Anatomy, Showa Univ. Sch. Med., Japan Dept. of Physical Educ., Hoshi Univ. Sch. Pharm. Pharm. Sci., Japan 3 Pharm. Res. Div., Takeda Chem. Ind. Ltd., Japan 2

Makiko Suwa, Masanori Arita, Kiyoshi ASAI and Yutaka Akiyama Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Japan We have developed an automated system for discovering GPCR genes in the whole human genome by integrating tools such as gene finding, sequence alignment, motif-domain assignment and transmembrane helix prediction, followed by gene quality refinement. The integration enabled us to detect multi-exon sequences and remote homologues that cannot be detected by conventional sequence search. By carefully assessing each component, we predicted GPCR genes in four confidence levels ranging from 827 candidates at the highest specificity to 2109 at the highest sensitivity. The overview of these datasets suggested that chromosome 11 has the largest number of GPCR most of which are olfactory/gustatory receptors and while chromosome 21 and Y shows the smallest number. It is interesting that the Chemokine receptors, one of the major families, are mainly concentrated on chromosome 3. These sequences are summarized in SEVENS database (http://sevens.cbrc.jp). We will also discuss about our approach to perform the structural and functional classification of these comprehensive candidates. The classification is based on not only sequence comparison but also on analyzing physico-chemical parameters.

Galanin-like peptide (GALP) is a novel 60 amino acid neuropeptide, isolated from porcine hypothalamus, which has been reported selectivity for the Gal-R2 galanin receptor. GALP mRNA was shown to be expressed predominantly in the rat hypothalamic arcuate nucleus (ARC), a region considered to be one of the most important feeding-regulating center in brain. Many GALP-containing neurons express leptin receptors and their mRNA is upregulated after treatment with leptin. However, it is reported that intracerebroventricular (i.c.v.) administration of GALP increases food intake in rats, thus GALP is assumed to be an orexigenic peptide. We have reported that neuropeptide Y (NPY)- and orexin-immunoreactive fibers are in close apposition with GALP-immunoreactive neurons. Furthermore, we have reported that the GALP-immunoreactive neurons express a-MSH and Orexin receptors in the ARC. The target neurons of GALP have not yet been identified. Orexin- and MCH-containing neurons are distributed in the LH; thus, we examined the relationship between GALP-containing neurons and orexin- and/or MCH-containing neurons in the LH by use of a dual immunostaining method. As a result, we found that GALP-immunoreactive nerve terminals were in apposition with Orexin- and MCHimmunoreactive cell bodies and their processes. These findings suggest that GALP-containing neurons make various complicated neuron networks in the hypothalamus and it may participate in the regulation of feeding behavior.