Twelfth Annual Summer Neuropeptide Conference, June 29–July 3, 2002, Marco Island, Florida, USA

Neuropeptides (2002) 36 (5), 371–386

SUMMER NEUROPEPTIDE CONFERENCE 2002 MEETING REPORT AND ABSTRACTS

doi:10.1016/S0143-4179(02)00049-5, available online at http://www.idealibrary.com on

Twelfth Annual Summer Neuropeptide Conference, June 29–July 3, 2002, Marco Island, Florida, USA T.W. Moody,1 I. Gozes2 1

Office of the Director, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA Department of Clinical Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel

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The 2002 Summer Neuropeptide Conference, which was held June 29–July 3, 2002 in Marco Island, Florida, USA, was attended by approximately 70 registrants from Brazil, Canada, France, Germany, India, Israel, Italy, Taiwan, the UK, and the USA. The annual meeting presented the latest discoveries in neuropeptide research. The meeting is organized independently by neuropeptide researchers from universities, research institutes, and pharmaceutical companies and are members of the nonprofit International Neuropeptide Society. This year’s meeting in Florida provided an opportunity for neuropeptide researchers to present their findings in formal symposia, poster presentations, and informal discussions. The symposium topics were neuropeptides and drug abuse, vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VIP/PACAP), neuropeptides and immune response, gastrointestinal (GI) peptides, nonpeptide antagonists, neuropeptide biotechnology, and neuropeptide polymorphisms. The keynote address was presented by Dr John Hughes, who discussed the sequencing of enkephalins. In addition, 17 presentations were made at the poster session. Eight fellows were provided travel awards to present their research at the meeting. The symposium on neuropeptides and drug abuse was chaired by Dr Z. Sarnyai (Psychogenics). Dr Sarnyai described the relationship of cocaine administration in rats and CRF receptors. Cocaine self-administration in rats is modulated by brain CRF receptors. Central CRF receptor blockade alleviates anxiety-like behavior associated with cocaine withdrawal. D. Adams (Yale Univ. Sch. Med.) demonstrated that chronic cocaine administration into rats increases melanocortin-4 receptors (MC4-R) in the striatum. Direct infusion of MC4-R antagonists into the Correspondence to: T. W. Moody, Office of the Director, Center for Cancer Research, Building 31 Room 3A34 31 Centre Drive, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Fax: 1-301-480-4323; E-mail: moodyt@mail. nih.gov

rat nucleus accumbens blocks cocaine induced locomotor sensitization, conditioned place preference, conditioned reward and self-administration. S. Bhalla (Univ. Illinois) found that BQ123, an endothelin-A (ETA) receptor antagonist, potentiated morphine analgesia and prevented adverse effects of morphine. Dr U. Shalev (Natl. Inst. Drug Abuse) found that food deprivation reinstates heroin and cocaine seeking behavior in rats and mice. Intraventricular administration of leptin, a hormone involved in energy balance and food intake, blocked this effect in heroin-trained rats. Dr M. Picciotto (Yale Univ. Sch. Med.) found that galanin attenuates the rat place preference conditioned by peripheral administration of morphine. Acute rat treatment with morphine or naloxone decreased and increased, respectively, galanin receptor binding in the nucleus accumbens. Dr F. Crespi (GlaxoSmithKline) demonstrated that cholecystokinin (CCK) receptor antagonists reduce spontaneous drug intake in ethanol drinking and cocaine drinking rats. The symposium on VIP/PACAP was chaired by Dr I. Gozes (Tel Aviv Univ.). Dr M. Laburthe (INSERM) identified amino acids of VIP that are essential for high affinity binding to VPAC1 and VPAC2 receptors. By site directed mutagenesis analysis of VPAC1 receptors, amino acids 322 and 394 were found to be essential for coupling of the receptor to adenylyl cyclase. Dr D. Brenneman (NIH) found that activity-dependent neurotrophic factor (ADNF) consists of a complex of three proteins which promote neuronal survival by stimulating protease activity. Dr I. Gozes reported that VIP increases ADNP expression. ADNP is expressed early in development (E7.5) and homozygous ADNP-knockouts die in the uterus. Dr N. Berman (Univ. Kansas Med. Ctr.) reported that CGRP is implicated in trigeminally mediated pain while estrogen modulates nociceptive or cerebrovascular responses, in part through NPY. Dr J. Waschek (UCLA) reported that upregulation of PACAP in motor neurons after injury is due to the presence

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of inflammatory cells at the injury site and loss of targetderived factors. Dr T. Moody (NIH) reported that PACAP but not VIP causes increased tyrosine phosphorylation of focal adhesion kinase and paxillin in human lung cancer cells. The signal transduction mechanisms of PACAP are more complex than those of VIP. Dr D. Ganea (Rutgers Univ.) chaired the session on neuropeptides and immune response. She found that VIP/ PACAP alter the Th2/Th1 balance by acting on both macrophages and CD4 T cells. VIP and PACAP promote Th2 survival by inhibition of Fas ligand expression. Dr E. Goetzl (UCSF) characterized immune cells derived from VPAC2 receptor transgenic animals. The survival advantage mediated by VPAC2 receptors is greater for Th2 than Th1 cells due to increased proliferation and decreased apoptosis. Dr J. Weinstock (Univ. Iowa) reported that interleukin (IL)12 and IL 18 but not IL10 act through a NFkB dependent pathway to induce expression of the NK-1 receptors on T cells. Dr P. Rameshwar (Rutgers Univ.) found that high levels of substance P (SP) precursor peptides are present in meloproliferative diseases (MPD). Also, NK-1 receptors may be involved in the proliferation of breast cancer cells. These results suggest that VPAC2 and NK-1 receptors are present on immune cells. Dr J. Pisegna (UCLA) chaired the session on gastrointestinal (GI) peptides. PAC1 receptor activation causes phosphatidylinositol (PI) turnover and elevates cAMP in ECL cells. Deletion of 63 amino acids of the C-terminal of the PAC1 receptors impairs PI turnover and elevation of intracellular cAMP. Dr Y. Tache (UCLA) found that corticotropin releasing factor (CRF)1 receptors regulate colonic defecatory and visceral pain in response to stress, whereas CRF2 receptors regulate a gastroprotective effect and reduce visceral pain to colorectal distension. Dr W. Chance (Univ. Cincinnati Med. Ctr.) found that glucagon-like peptide-2 (GLP-2) is growth-promoting in the proximal intestine if sufficient polyamines (putrescine and spermidine) are present. Dr N. Bunnett (UCSF) reported that NK-1 receptors sequester b-arrestins in endosomes, depleting cytosolic pools and preventing b-arrestin-dependent endocytosis and desensitization of the NK-3 receptor. The keynote address was delivered by Dr J. Hughes (Univ. Cambridge). He discussed the sequencing of methionine- and leucine-enkephalin when he was at the University of Aberdeen. Also, he discussed the synthesis of nonpeptide cholecystokinin receptor antagonists when he was at the Parke-Davis Research Centre. The session on nonpeptide receptor antagonists was chaired by Dr J. Hughes. Dr A. McKnight (Oxford Natural Products) described the development of NK-1 nonpeptide receptor antagonists. CP-96,345 may be useful as an antidepressant and/or anti-emetic. Dr S. Dax (Johnson & Johnson) described aminotetralin analogues as Y5 receptor antagonists Dr C. Gerald (Synaptic Pharmaceuticals) Neuropeptides (2002) 36(5), 371–386

discussed the development of ligands for orphan receptors. Also, the MCH-1 receptor antagonist SNAP 7941 was investigated as a anti-anxiety or anti-depressant agent. Dr M. Webb (Pharmacopieia) characterized bradykinin (BK) antagonists. PS309799 binds with high affinity to B1 receptors and antagonizes the effect of des-Arg-kallidin to cause hypotension and pro-inflammatory edema. The session on peptide biotechnology was chaired by Dr I. Gozes. Dr R. Bohrer (Cal. Western Sch. Law) emphasized that requirements to patent neuropeptide receptor sequences are changing and require additional information about protein biological function. Also, genomics and proteomics are becoming an increasingly important research and patent areas. Dr S. McLean discussed using microarray data for identifying new neuropeptide orphan receptors. Dr A. Reitz (Johnson & Johnson) described the role of recombinant proteins such As Procrit and Remicade for therapeutic use. Dr I. Antal (Bristol-Myers Squibb) discussed neuropeptides involved in obesity. NPY is well known for its ability to stimulate food intake in rats, whereas numerous neuropeptides such as bombesin and CCK inhibit food intake. Recently, ghrelin was shown to be important in weight loss in humans. The session of peptide polymorphisms was chaired by Dr J. Quinn (Univ. Liverpool). Dr C. Baerwald (Univ. Clinic Leipzig) identified two biallelic polymorphic sequences for CRF in rheumatoid arthritis (RA) patients. CRF promoter polymorphisms may represent new genetic marker for RA susceptibility. Dr F. Grant (Harvard Med. Sch.) has identified over 30 polymorphisms in the vasopressin (VP) gene in familial diabetes insipidus (FDI) patients. Some of the mutations impair the ability of the kidney to concentrate urine. Dr J. Quinn discussed the variable number of tandem repeats (VNTR) within intron 2 of the serotonin transporter gene, and the 30 untranslated region of the dopamine transporter gene. It remains to be determined if VNTR polymorphisms are associated with behavioral disorders. The application of all innovative ideas presented in the various symposium to human therapy still needs to be elucidated. Seventeen excellent poster presentations contributed to the breadth and depth of the scientific presentations. At the conference dinner, Dr K. Seroogy (Univ. Kentucky) presented awards to the eight winners of Graduate Student and Postdoctoral Fellow Travel Awards. The beach location of the conference Marco Island Marriott combined with recreational attractions of Marco Island enhanced the informal scientific interactions. The Thirteenth Annual Conference will be a joint meeting of the Summer Neuropeptide Conference and the European Neuropeptide Club, to be held in Montauk, New York, USA, June 8–12, 2003 (contact email: igozes@ post.tau.ac.il). The abstracts for the Twelfth Annual Summer Neuropeptide conference are provided herein.

Twelfth Annual Summer Neuropeptide Conference Abstracts

ABSTRACTS Corticotropin-releasing factor (CRF) and cocaine addiction Zolta´ n Sarnyai, PsychoGenics Inc. and Rockefeller University. CRF plays a major role in stress response and may have physiological significance in the development and maintenance of and relapse to drug addiction. CRF mediates the neuroendocrine and some of the acute behavioral effects of cocaine in rats, rhesus monkeys and in humans. CRF-driven activation of the hypothalamic-pituitary-adrenal (HPA) axis plays an important role in psychostimulant-induced behavioral sensitization. Cocaine selfadministration in rats has been shown to be sensitive to modulation of brain CRF receptors. Long-term cocaine administration results in alterations in hippocampal CRF receptors. Moreover, withdrawal from chronic cocaine administration leads to increased CRF activity in several limbic-forebrain structures, most importantly in the amygdala. Central CRF blockade alleviates anxiety-like behavior associated with cocaine withdrawal. Furthermore, CRF in the amygdala-bed nucleus of stria terminalis system has been shown to play a key role in stress-induced relapse to cocaine self-administration. These data suggest that extrahypothalamic brain CRF systems are critically involved in drug withdrawal and relapse to drug-taking induced by stressors. On the other hand, it appears that hypothalamic CRF, via its action on the HPA axis, is involved in the locomotor activating and reinforcing effects of cocaine. These preclinical data may provide a rationale for the development of CRF-based pharmacotherapies for the treatment of compulsive cocaine use in humans.

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Morphine still remains the major drug for the treatment of cancer pain, however, excessive adverse effects limits it’s use. We provide evidence that BQ123, an endothelin-A receptor (ETA) antagonist, could be useful in potentiating analgesic and preventing the adverse effects of morphine. The effect of BQ123 (10 lg, i.c.v.) on morphine-induced analgesia, hyperthermia, and catalepsy was determined in the rat. Morphine (8 mg/kg, s.c.) produced a significant increase in tail flick latency as compared to control group (Figure 1). BQ123 significantly potentiated the analgesic effect of morphine as compared to vehicle-treated control rats. The hyperthermic effect of morphine was also significantly potentiated by BQ123. BQ123 did not affect morphine-induced cataleptic behavior. The effect of BQ123 (3 lg, i.c.v.) on morphine analgesia and change in body temperature was determined in mice. Morphine (2, 4, and 8, mg/ kg, s.c.) produced a significant increase in tail flick latency that lasted for 1.5 hours. BQ123 significantly potentiated morphine analgesia, which lasted for more than 4 hours (Figure 2). BQ123 did not potentiate the hypothermic effect of morphine. The effect of BQ123 (3 lg, i.c.v.) on naloxone-precipitated moiphine withdrawal was also determined in nice. Naloxone (1 mg/kg, i.p.) administration to morphine tolerant mice (1 morphine 75 mg pellet for 3 days) resulted in expression of withdrawal symptoms. BQ123 did not affect hypothermia, loss of body weight, jumping behavior, falls, diarrhea, fecal boli, urination, ptosis, writhing, and rearing behavior during withdrawal. Studies were also carried out to determine tolerance to analgesic effect in morphine tolerant dependent rats (6 morphine pellets in 7 day period). BQ123 (10 lg, i.c.v., twice a day for 7 days) treated rats did not

Role of melanocortins in drug addiction Ronald S. Duman, Jane Taylor, David Adams, Richard Hsu, Yale University School of Medicine. Studies from our laboratory demonstrate that chronic administration of cocaine increases the expression of the melanocortin-4 receptor (MCR-4) in rat striatum. These findings raise the possibility that melanocortins and MC4-R are involved in the longterm changes underlying the behavioral actions of cocaine, and possibly other drugs of abuse. To test this possibility we have used a combination of pharmacological and mutant mouse approaches to test the role of the melanocortin-MC4-R system in behavioral models of cocaine action. Direct infusion of a melanocortin antagonist into the nucleus accumbens blocks several cocaine-induced behaviors, including locomotor sensitization, conditioned place preference, conditioned reward, and self-administration. In addition, cocaine-induced locomotor sensitization was completely blocked in MC4-R null mutant mice indicating that this melanocortin receptor subtype is necessary for the actions of cocaine in this behavioral paradigm. In addition, co-localization studies demonstrate that the MC4-R is colocalized with dynorphin-, but not enkephalin-, expressing neurons, in the nucleus accumbens, suggesting that interactions of MC4-R with Dl-dopamine receptors is necessary for the rewarding actions of cocaine. The results also indicate that an antagonist of MC4-R could be efficacious in the treatment of drug abuse in humans. BQ123 potentiates morphine analgesia, prevents morphine tolerance to analgesia and does not affect naloxone-precipitated withdrawal: a novel approach for cancer pain Anil Gulati, Shaifali Bhalla, George Matwyshyn, Department of Pharmaceutics & Pharmacodynamics, The University of Illinois at Chicago, Chicago, IL 60612, USA.

Fig. 1 Effect of BQ123 on morphine (8 mg/kg) analgesia in rats.

Fig. 2 Effect of BQ123 on morphine (2 mg/kg) analgesia in mice. Neuropeptides (2002) 36(5), 371–386

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become tolerant to morphine. These studies are the first to demonstrate that BQ123 potentiates analgesic effect of morphine, prevents the development of tolerance to morphine analgesia, without affecting naloxone-precipitated morphine withdrawal. Conclusion: A combination use of BQ123 and morphine could provide a novel approach in improving the analgesia and reducing morphine adverse effects and may be useful in the treatment of cancer and other types of pain.

Relapse to drugs and control of eating: are the underlying brain mechanisms related? Provocative results with food deprivation and leptin Uri Shalev, NIDA, Intramural Research Program, National Institute of Health. Studies in humans suggest that exposure to stressors provokes relapse to drug use. The mechanisms underlying stress-induced relapse, however, are not known. We use the reinstatement model to study behavioral and neurochemical substrates that mediate the phenomenon of stress-induced relapse to heroin and cocaine seeking in rats. In this model, the effect of acute exposure to drug or non-drug stimuli on reinstatement of drug seeking is examined after training for intravenous (IV) selfadministration of drugs and subsequent extinction of the drug-reinforced behavior. Using this model it was shown that re-exposure to the self-administered drug or exposure to a footshock stressor, induce reinstatement of drug seeking. More recently, we have shown that acute, 1-day food deprivation potently reinstates heroin and cocaine seeking in rats and mice. Intraventricular administration of leptin, a hormone involved in energy balance and food intake, blocked this effect in herointrained rats, but did not affect footshock- or drug-induced reinstatement. In addition, corticosterone, a major stress hormone, was shown to have a permissive role in food deprivation-induced reinstatement of cocaine, but not heroin, seeking. These data suggest that food deprivation can provoke relapse to heroin seeking via a leptin-dependent mechanism, which is not involved in relapse induced by footshock stress or re-exposure to heroin.

Galanin-opiate interactions in the CNS M. R. Picciotto, V. Zachariou, Dept. of Psychiatry, Yale University School of Medicine. The neuropeptide galanin potentiates morphine analgesia in spinal cord, but less is known about galanin-opiate interactions in the CNS. Galanin activates a family of G protein-coupled receptors. GalR1 and GalR2 are expressed in brain areas associated with drug addiction, such as the mesolimbic dopamine system and the locus coeruleus (LC). We found that galanin attenuates the place preference conditioned by peripheral administration of morphine. This regimen of morphine treatment decreased galanin receptor binding in the nucleus accumbens (NAc) and increased galanin binding in the LC. In contrast, acute naltrexone administration increased galanin binding in the NAc, suggesting that levels of galanin receptors are tonically regulated by opioid receptors in that area. Galanin binding in LC was also upregulated by chronic-intermittent morphine administration or by precipitated withdrawal, but not by acute morphine treatment, suggesting that increased activity of LC neurons may regulate levels of galanin binding sites. Moreover, morphine withdrawal increased GalR1 mRNA in the LC, suggesting that regulation occurred at the level of transcription. We have also shown that a Neuropeptides (2002) 36(5), 371–386

galanin agonist can antagonize morphine withdrawal signs. We hypothesize that the galanin system in the brain is normally protective against the development of opiate dependence and withdrawal.

CCK and craving: potential therapeutic role of CCK receptor antagonists Francesco Crespi, PhD, Biology Dept., Psychiatry-CEDD, GlaxoSmithKline S.p.A., Medicines Research Centre, via Fleming 4, Verona, Italy. Original approaches to study drug craving and the influence of CCK receptor antagonists upon this state will be presented. Briefly: (1) a free-choice model could allow naive rats to reveal an innate tendency to consume drugs such as cocaine or alcohol, and therefore the study of the individual tendency toward drugs of abuse. (2) An original behavioral model for craving allows the combination of behavioral observations together with electro-biochemical (voltammetric) analysis of neurotransmitters such as catechols, 5-OH indoles and peptides in conscious freely moving rats, in situ, in discrete brain regions(s) and in real time. The data gathered in these experiments may indicate that not only the dopaminergic system, but also central peptidergic functions could play a major role within the drug dependency phenomenon. Further analyses are required to fully define the chemical nature of in vivo DP Voltammetric Peak 5. However, it is possible to conceive that the signal recorded in the nAccumbens is related to the oxidation of a CCK-like compound, and the related results give rise to the original hypothesis of the involvement of peptidergic activities in the modulation of the drug seeking (craving) state. This hypothesis is supported by pharmacological evidence that CCK receptor antagonists reduce spontaneous drug intake in ethanol drinking and cocaine drinking rats. These data suggest such compounds as a novel putative therapeutic approach for drug craving.

VPAC receptors: molecular pharmacology and structure Marc Laburthe, INSERM U410, Neuroendocrinology and Cell Biology, Fac. Bichat, 75018 Paris, France. VIP biological actions are mediated by two receptors VPAC1 and VPAC2 belonging to the class II family of G protein-coupled receptors. VPAC receptors trigger biological responses through cAMP production though other signaling pathways have been described. Recent studies of the human VPAC1 receptor documented the structure-function relationship of VPAC receptors. The following data will be presented: (1) Site-directed mutagenesis and a 3D model of the N-terminal extracellular domain of the VPAC1 receptor which plays a predominant role in VIP recognition; (2) Circumstancial evidence for a second binding domain on the core of VPAC1 receptor in agreement with theories of class II receptor activation; (3) Complete alanine scanning of VIP: aminoacid residues participating in VIP binding to VPAC receptor were identified. This leaded to the design of a selective VPAC1 receptor agonist e.g. [Ala 11,22,28]VIP; (4) Important aminoacid residues of VPAC1 receptor for coupling to adenylyl cyclase are present in intracellular loop (IL) 2, IL 3 and C-terminal tail (C-tt). Construction of the mutant K322A(IL3)/E394A(C-tt) results in a dramatic reduction of coupling. A rhodopsin-based structural model of the core of VPAC1 receptor provides evidence for an intracellular 3Ddomain responsible for coupling to adenylyl cyclase.

Twelfth Annual Summer Neuropeptide Conference Abstracts

Functional and structural characteristics of the activitydependent neurotrophic factor complex: a mediator of VIP action in the central nervous system D. E. Brenneman1, J. Hauser1, R. Castellon1, Y. Zhao1, Y. Li1, C. Y. Spong2, I. Gozes3, 1Section on Developmental and Molecular Pharmacol., NICHD; 2Pregnancy and Perinatology Branch, NICHD; and 3Clinical Biochem., Sackler Med. Sch., Tel Aviv Univ., Israel. Vasoactive intestinal peptide (VIP) regulates developmental processes in the central nervous system including neuronal apoptosis, excitatory synaptogenesis, neurite extension, and mitosis. Many of these actions are mediated indirectly through the release of astrocyte-derived proteins, including activity-dependent neurotrophic factor (ADNF). In the present study, ADNF was shown to exist as a complex of three components. The ADNF components were identified by three distinct, survivalpromoting peaks isolated by N-CHO capillary electrophoresis. The components varied greatly from each other in their neuroprotective potency against tetrodotoxin (8 orders of magnitdue). Furthermore, each was neutralized by a different group of antibodies directed against digest peptides derived from ADNF. The digest peptides exhibited low sequence homology to known proteases. Importantly, treatment with a cocktail of protease inhibitors prevented all survival-promoting activity of ADNF preparations. ADNF components exhibited different responses to specific serine, aspartyl and cysteine protease inhibitors. These data suggest that ADNF exists as a complex and produces survival-promoting effects through multiple proteases intrinsic to or interactive with three protein components.

Neuropeptide cyclicity in trigeminal ganglia from intact female mice Nancy E. J. Berman1, Lisa Cui1, Veena Puri1, K. Michael A. Welch2, Robert M. Klein1, 1Department of Anatomy & Cell Biology; 2Department of Neurology; and 1,2Migraine Research Laboratory, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7400. Migraine and temporomandibular joint (TMJ) pain are both linked to the menstrual cycle. One mechanism may involve ovarian steroid regulation of neuropeptide synthesis by neurons in the trigeminal ganglia. Our focus is on selective peptide neurotransmitter systems involved in pain and cerebrovascular control, calcitonin-gene related peptide (CGRP), atrial natriuretic peptide (ANP) and neuropeptide Y (NPY). Gene expression was analyzed in the trigeminal ganglia of cycling female mice using reverse transcription polymerase chain reaction. Stages of the estrous cycle were identified by vaginal smears. Expression of CGRP did not change across the estrous cycle. Expression of NPY was strongly linked to the estrous cycle, peaking at estrus. Expression of ANP was only weakly linked to the estrous cycle. Our findings are compatible with CGRP being directly implicated in trigeminallymediated pain activation, while estrogen modulates nociceptive or cerebrovascular responses, in part through effects on NPY. The effects of estrogen on neuropeptide gene expression may explain female susceptibility to trigeminally-mediated headache and facial pain. Supported by the American Heart Association Heartland Affiliate and NIH grant P50NS32399. Activity-dependent neuroprotective protein (ADNP): a VIPresponsive regulator of embryonic development I. Gozes1, A. Pinhasov1, E. Giladi1, M. Bassan1, A. M. Goldsweig1, S. Servoss2, A. Grinberg3, H. Westphal3 & D. E. Brenneman2,

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Clinical Biochem. Sackler Med. Sch., Tel Aviv Univ. Israel; SDMP, LDN; and 3LMGD, NICHD, NIH, Bethesda, MD, USA.

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A novel vasoactive intestinal peptide (VIP) responsive gene, ADNP, was recently cloned (J. Neurochem. 72, 1283, 1999). Results have indicated that this gene is associated with neuroprotection and cell proliferation. Inhibition of ADNP synthesis by antisense oligodeoxynucleotides resulted in cancer cell death (J. Bio. Chem. 276, 708, 2001). To further assess ADNP’s role in cell division and growth, we have taken two complementary approaches. (1) Measurements of ADNP gene expression during mouse development by Northern blot hybridization; and (2) establishment of ADNP gene knockout mice. Results showed that (1) the embryonic ADNP gene was already expressed during early gestation, detected on E7.5 and peaked on E9.5. Expression was increased in the brain (E12) and was sustained throughout embryogenesis. (2) A BAC clone containing the mouse ADNP gene was mapped. Appropriate 5 and 3 gene flanking Fragments were chosen for homologous recombination and were sub cloned into a vector containing a neomycin-resistance coding site. Stem cells that underwent homologous recombination were selected and chimeric mice were obtained. Further breeding and Southern blot analysis indicated that homozygous ADNP-knockouts die in the uterus. Macroscopic analysis revealed cranial neural tube closure failure manifested in anencephaly and death on E8.5-9.0. Incubation of E8.5 mouse embryos with VIP results in robust embryonic growth (Nature 362,155, 1993) as well as in increased ADNP gene expression. Thus, ADNP may mediate the VIP-stimulated embryonic growth while serving a vital role in brain maturation. (Supported by BSF, Neufeld, ISF, Gildor, ISOA, NIH.)

PACAP action in nervous system development and Regeneration J. Waschek, V. Lelievre, Z. Hu, W. Rodriguez, J. Cheng, D. Chun, M. Yamamoto, A. Flores, Department of Psychiatry and Mental Retardation Research Center, Neuropsychiatric Institute, University of California at Los Angeles, Los Angeles, CA. PACAP and its receptors are expressed in the developing brain beginning at the onset of neurogenesis. We and others have shown that PACAP regulates several aspects of neuronal, astrocyte and oligodendrocyte progenitor development in vitro. These include proliferation, survival, neurite outgrowth, myelinogenesis, and expression of trophic factors, cytokines, and their receptors. We have determined that the actions of PACAP can be substantially modified, and in some cases completely reversed by the presence of other growth factors such as sonic hedgehog, FGF-2, and PDGF. This suggests that in vivo actions of PACAP might vary widely in different parts of the brain and at different phases of development. PACAP is also strongly upregulated after nerve injury in adult animals, and may therefore regulate the injury response. Our data suggest that the upregulation of PACAP in motor neurons after injury is due to (1) the presence of inflammatory cells at the injury site and (2) a loss of target-derived factors. Mice with a targeted mutation on the PACAP gene have been generated to study the developmental and injury actions of this peptide.

PACAP causes focal adhesion kinase tyrosine phosphorylation in lung cancer cells T. W. Moody, J. Leyton, L. Garcia, R. T. Jensen, NCI Office of the Director, Center for Cancer Research, Bethesda, MD and Digestive Diseases Branch, NIDDK, Bethesda, MD 20892. Neuropeptides (2002) 36(5), 371–386

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Addition of PACAP-27 to lung cancer cells, causes increased cAMP and PI turnover. Previously, we found that addition of bombesin-like peptides to lung cancer cells increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin (Leyton et al., Cancer Lett. 162:87(2001)). Here the effects of PACAP on FAK tyrosine phosphorylation were investigated. PACAP-27 caused FAK tyrosine phosphorylation after addition to NCI-H1299 cells which was maximal using 100 nM PACAP27 for 2.5 min. PACAP-38 but not PACAP (6–38) or PACAP (28–38) caused FAK tyrosine phosphorylation after addition to NCI-H1299 cells. PACAP (6–38), however, blocked the increase in FAK tyrosine phosphorylation caused by PACAP-27 or PACAP-38. Also, genistein, H7 and cytochalasin D, which are tyrosine kinase inhibitors, serine/threonine kinase inhibitors and actin polymerization inhibitors, reversed the tyrosine phosphorylation of FAK caused by PACAP. It remains to be determined if FAK regulates lung cancer proliferation and/or metastasis.

VIP and PACAP promote Th2-type immune responses Doina Ganea1, Mario Delgado1,2, Rosa Rodriguez1, 1Rutgers University, Newark, NJ; and 2Universidad Complutense, Madrid, Spain. In an immune response, antigen-specific CD4 T cells proliferate and differentiate into Th1 and Th2 effector cells, with different roles in cell mediated- and humoral immunity. Although both Th1 and Th2 effectors are normally generated, certain sites such as brain, anterior chamber of the eye, testis, and the intestine-associated Peyer’s Patches show a definite Th2 bias. These sites are also rich in VIP and PACAP. We proposed that the neuropeptides play a role in the Th2 bias. Here we present evidence that VIP and PACAP affect the Th2/ Th1 balance by acting on both macrophages and CD4 T cells. The major mechanism for the Th2-induced bias by macrophages involves inhibition of IL-12 production by VIP/PACAP. In addition, VIP and PACAP promote the short- and long-term survival of antigen-specific Th2, but not Th1 cells, both in vivo and in vitro. One of the mechanisms involved is the inhibition of Fas ligand expression specifically in Th2 cells. The fact that VIP and PACAP promote Th2-type responses is in general agreement with their anti-inflammatory activity, and is biologically significant since organs highly susceptible to local inflammation still need a functional immune response to pathogens.

Enhancement of survival of CD4 (helper-inducer, Th) T cells and preferential generation of Th2 memory cells by the vasoactive intestinal peptide (VIP) – Type II VIP receptor (VPAC2R) axis Edward J. Goetzl, Glenn Dorsam, Yvonne Kong, Carola Grinninger, Julia K. Voice, Depts. of Medicine and Immunology, UC Med. Cntr., San Francisco, CA 94143-0711. VIP and its G protein-coupled Rs in Tcells, constitutive VPAC1 and inducible VPAC2, are highly-functional in immunity. The effects of each VPACR on survival and differentiation of Th cells were studied in immunomag- netic bead-purified splenic CD4 T cells from T cell-targeted VPAC2 transgenic (TG) (FASEB J. 15:2489, 2001) and VPAC2-null (KO) (PNAS 98:13854, 2001) C57BL/6 mice, with equal VPAC1. CD4 cells were incubated with anti-CD3 + -CD28 antibodies (Abs) under Th1- and Th2-directing conditions (days 1-5), IL-2 alone Neuropeptides (2002) 36(5), 371–386

(d 6–7), and anti-CD3 Abs (d 8–9). Relative mean survival of TG:KO Th1 cells was 3.4 and 1.7 on days 7 and 9, respectively, and of Th2 cells was 3.8 and 7.0. Mean apoptosis (nucleosome ELISA) on days 7 and 9 was 3- and 6-fold higher for KO than TG Th1 cells and 3- and 4-fold higher for KO than TG Th2 cells. Mean proliferation (3H-thymidine uptake) on day 9 was 2-fold higher for TG than KO Th1 cells and 5-fold higher for Th2 cells. Intraperitoneal Staphylococcal enterotoxin A (SEA) superantigen elicited significantly more splenic Th2 memory cells in TG than KO mice 60 days later, as assessed by quantifying defining surface markers and cytokine profiles of CD4 T cells. The survival advantage mediated by VIP-VPAC2 is greater for Th2 than Th1 cells, due to both increased proliferation and decreased apoptosis, and contributes to heightened generation of Th2 memory cells. (Supported by A129912.)

The importance of substance P in regulation of chronic liver and intestinal inflammation Joel V. Weinstock, MD, Division of Gastroenterology, University of Iowa, Iowa City, Iowa 52242. Substance P (SP) is an immunoregulatory neuropeptide produced at sites of inflammation. Murine schistosomiasis is a parasitic disease in which parasite eggs induce focal, chronic granulomatous inflammation in the liver. Also, IL10 KO mice spontaneously develop chronic Th1-type intestinal inflammation resulting from the loss of IL10. In these two distinct models of chronic inflammation, SP is an inducible product of the leukocytes that infiltrate the liver and intestines. SP functions to substantially augment IFNc production and the Th1 response. The effect of SP on IFNc secretion is mediated by direct interaction with a SP receptor (NK1) expressed on the T cells. IL12 and IL18 are released innately shortly after immune stimulation to help trigger IFNc synthesis and Th1 cell development. IL12 and IL18 acting through the NFkB pathway induce expression of the SP receptor on T cells. IL10, an anti-inflammatory cytokine, can prevent or down-modulate T cell NK1 receptor expression. Development of T cell-selective NK1 KO mice has demonstrated the critical role of this inducible SP receptor on T cells for control of inflammatory responses.

Therapeutic potential of a-MSH: present and future perspectives Anna Catania, Gualtiero Colombo, Stefano Gatti, James M. Lipton*, Ospedale Maggiore di Milano IRCCS, 20122 Milano, Italy; * Zengen Inc, Woodland Hills, CA 91367, USA. a-MSH (a-melanocyte stimulating hormone), a melanocortin peptide, is known for its neuroimmunomodulatory properties. This molecule serves as an endogenous mediator in the brain, pituitary, circulation, peripheral tissues, and host cells in modulation of production and actions of proinflammatory agents. Exogenous a-MSH modulates inflammatory responses in animal models. Furthermore, it is increased in clinical inflammatory disease in humans, presumably as a natural countermeasure to inflammation. The tridecapeptide a-MSH and its COOH-terminal tripeptide amino acid sequence KPV (a-MSH 11–13) have parallel anti-inflammatory effects in animal models and in in vitro tests on host cells. There is recent evidence that these peptides also have antimicrobial properties that can benefit the host.

Twelfth Annual Summer Neuropeptide Conference Abstracts

Neurokinin (NK) receptors as potential targets for drug development in stem cell disorders of the bone marrow (BM) K. Sancilio, J. Potian, S. Greco, S. Won, P. Bandari, P. Rameshwar, UMDNJ-NJMS. Immune cell development occurs by the process of hematopoiesis in the adult BM. This organ has two types of stem cells that support the functions of each other: (1). Lymphohematopoietic (LHSC), which are found in the area with the lowest oxygen level and, (2). Mesenchymal (MSC), which surrounds the vasculature. Differentiated MSC (fibroblasts) support the LHSC at the different levels of the hematopoietic hierarchy. The NK receptors and the gene that encodes their ligands (PPT-I) interact with different families of growth factors and neurotransmitters to regulate stem cell functions. The NK receptor, PPT-I ligands and other molecules that are similar to NK-1, are linked to disruption of the BM stem cell compartment. The experimental evidence identifies two major types of stem cell disorders. I. Myeloproliferarive diseases (MPD): Dysregulation occurs at two levels: proliferation of the myeloid clone and expansion and differentiation of the MSC. In MPD, PPT-I peptides evade digestion from endogenous endopeptidases by interacting with increased levels of fibronectin. II. Stem cell disruption by endocrine cancer in the BM: Gene silencing of PPT-I or NK-1 by RNAi in breast cancer cells blunted their integration in the BM. While NK-1 seems to be involved in the proliferation of cancer cells, NK-2 might be involved in the protection of the ‘cancer stem cells’ in the BM. The experimental evidence indicates that the NK receptor could be drug targets for MPD and breast cancer cells in the BM.

Pharmacology, signal transduction, localization and function of PACAP and PAC1 Joseph R. Pisegna, CURE/VA Greater Los Angeles Healthcare System and Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA. The receptor for Pituitary Adenylate Cyclase Activating Polypeptide (PAC 1) is a heptahelical, G protein-coupled receptor (GPCR) is coupled to the activation of both inositol phosphates and adenylyl cyclase (AC) and to the activation of immediate early genes in transfected cells. PAC 1 has been shown to be localized to the gastric and colonic mucosa where they exert their physiological effects. Similarly in cultured tumoral cell lines, PACAP has been shown to be a potent growth factor in neuronal and tumoral cells and as a mediator of nocturnal gastric acid secretion. We have previously demonstrated that PAC 1 receptors are expressed on gastric ECL cells leading to the activation of [Ca++]i, histamine release and to gastric acid secretion. The regions of the PAC1 receptor important for signal transduction and internalization have been characterized on the C-terminus of the receptor. Truncation of the entire 63 a.a. of the hPAC1 resulted in no signaling to either AC or IP whereas, addition of the proximal 27 a.a. of the C-terminus resulted in reconstitution of complete AC and IP responses identical to the wildtype (EC50 = 0.6–1.5 nM). Point mutations within this 17 a.a. region identified specific amino acids involved in PAC1 signaling indicating that a structural motif within the proximal region of carboxyl terminus is critical for G protein-coupling. In addition, the t1/2 for internalization of ligand for the truncated mutants were increased to 6.7–9.6 min. compared to 3.7–4 min for the wild type receptor. Studies on rat ECL cells (90% pure) were performed by isolation and purification by elutriation, density centrifugation. More recently,

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FACS analysis permits 99% purity of these cells using a fluorescently tagged PACAP molecule. PACAP-27 dose-dependently stimulated ECL cell adenylyl cyclase (’6-fold increase at 100 nM). In isolated, superfused gastric glands following administration of PACAP-38, there was an increase in [Ca++]i in the ECL cells followed by calcium stimulation in the adjacent parietal cell at approximately 100 msec. Co-administration of an H2 receptor antagonist had no effect on the stimulation of [Ca++]i in the ECL cells, but effectively blocked stimulation of the parietal cell. Chronic administration of PACAP-38 (10 pmol/ hr for 7d) via osmotic pump in the rat resulted in greater than a two-fold increase in BrdU incorporation into ECL cells. These results indicate that the distal region of carboxyl terminus of the hPAC1 is involved with ligand-activated internalization of the hPAC1 and that a structural motif within the proximal region of carboxyl terminus is important in G protein coupling. PACAP acting at the PAC 1 results in dual signaling responses to both [Ca++]i and AC in ECL cells, stimulates gastric acid secretion via the actions of histamine acting at the parietal cell and in whole animals leads to proliferation of ECL cells when administered chronically. PACAP and its receptor, PAC1 are colocalized in gastric and colonic epithelium to modulate physiological function. These results provides convincing evidence that the neuropeptide, PACAP, produces signals in the GI tract in a manner consistent with that in transfected cell systems and is not only a potent regulator of gastric acid secretion but also is a neuropeptide involved in regulating the proliferation of ECL cells.

Dual actions of CRF receptor activation on gastrocolonic function in rodents Y. Tache´ , M. Million, L. Wang, V. Martinez, K. Kanamoto, CURE/ Dig. Dis. Res. Ctr., UCLA Dept of Medicine and VA Health Care System, Los Angeles, CA 90073, USA. Ucorcortin (Ucn) II and Ucn III are new members of the CRF family that bind selectively to CRF2 receptor. Intracerebroventricular (icv) injection of CRF inhibited gastric emptying while stimulating distal colonic transit and the gastrocolic responses were blocked by CRF2 and CRF1 receptor antagonists respectively in mice. The CRF-related peptides oCRF and rUcn injected icv also stimulated pellet output in mice. Restraint (60 min) induced fecal pellet output which is completely prevented by the CRF1 antagonist, NBI-458, and unmodified by astressin2-B in mice. Activation of central CRF1 receptors is also involved in the activation of locus coeruleus neurons to colorectal distention and mediate stress-related increased visceral pain in rats. Peripheral injection of Ucn II and to a lesser degree Ucn III inhibited gastric emptying of a solid meal, while not influencing distal colonic transit in mice and rats. Peptide actions were reversed by the peripheral injection of selective CRF2 antagonists, antisauvagine or astressin2-B while selective CRF1 antigonists had no effects. In rats, Ucn II increased gastric mucosal blood flow and prevented ethanolinduced gastric erosions through CRF2 activation and nitric oxide independent pathways. Ucn II injected iv prevented colorectal distention induced visceral pain through activation of CRF2 receptors. These data indicate that central subtype 1, mediates the colonic defecatory and visceral pain responses to stress while peripheral activation of CRF2 receptors exerts a gastroprotective effect and reduced visceral pain to colorectal distention. The dual actions of CRF1 and CRF2 receptors may represent adaptive responses to maintain homeostasis during stress. Neuropeptides (2002) 36(5), 371–386

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Polyamines are required for glucagon-like peptide-2-induced gut hyperplasia William Chance, PhD, S. Sheriff, PhD, A. Balasubramaniam, PhD, Cincinnati VA Med Ctr, Shriners Hospital for Children, and Dept Surgery, Univ Cincinnati Med Ctr, Cincinnati, OH. Glucagon-like peptide-2 (GLP-2) is released from the L cells of the gut upon feeding, and appears to dramatically influence maintenance of small intestine mucosal epithelium. Thus, coinfusion of GLP-2 with parenteral nutrition (PN) totally prevented depletion of small intestine mucosa in rats. We tested the hypothesis of polyamine involvement in GLP-2-induced hyperplasia using difluoromethylornithine (DFMO; gift from C. Levenson, PhD, Ilex Oncology) to block ornithine decarboxylase (ODC) and deplete polyamines in GLP-2-treated, male, Fischer 344 rats, maintained on PN. Normal saline was infused (2.0 ml/h) into catheterized rats for three days, after which they were switched to PN (2.5 ml/h, isotonic and isocaloric to rat chow; 1.1 kcal/ml) for seven to eight days. In each study, GLP2 was co-infused with PN in two groups at a concentration of 0.5 ug/ml, and one of these groups also received DFMO, iv (1st study: 5 mg/ml; 2nd study 10 mg/ml). The rats tolerated these treatments well, with no signs of distress being observed. At euthanization, protein was decreased significantly by PN in duodenum (by 25%), jejunum (by 46%) and ileum (by 44%), and restored by GLP-2 (duodenum = 135%, jejunum = 103%; ileum = 81%, compared to chow-fed control)). The colon was depleted by PN but was not affected by the GLP-2 treatment. Addition of DFMO to PN reduced the protective effect of GLP-2 in duodenum and jejunum by 34%, but not in ileum (7% reduction compared to GLP-2 PN). Assessment of jejunal and ileal polyamine concentrations revealed levels of putrescine and spermidine reduced by nearly 70% in DFMO-treated rats, while spermine was not affected. These results suggest that normal levels of the polyamines, putrescine and spermidine, are necessary for GLP-2 to express its growth-promoting effect in the proximal intestine mucosa. In addition, in the more distal gut GLP-2 was not as potent, and the observed hyperplasia appeared to be less dependent upon normal levels of polyamines.

Mechanism, function and control of agonist-Induced trafficking of neurokinin receptors Nigel Bunnett, PhD, Departments of Surgery and Physiology, University of California, San Francisco, USA. Agonists induce endocytosis of many G-protein coupled receptors, including neurokinin receptors (NKRs) To evaluate the mechanism of this trafficking, we expressed dominant negative mutants of proteins that regulate vesicular transport. Substance P (SP) induced membrane translocation of b-arrestin 1 and 2 followed by endocytosis of b-arrestins and NK1R into the same endosomes containing the GTPase rab5a, determined by confocal microscopy. Dominant negative b-arrestins, dynamin and rab5a inhibited NK1R endocytosis, and dominant negative rab5a also impaired trafficking of NK1R endosomes to a perinuclear region. To assess the function of trafficking, we measured MAP kinase activation and desensitization and resensitization of Ca2+ mobilization. SP activated the MAP kinases ERK1 and 2. Dominant negative b-arrestins, but not dynamin or rab5a, inhibited this activation. A truncated form of the NK1R (NK1Rd325), corresponding to a natural variant, was unable to associate with b-arrestins and exhibited diminished endocytosis and ERK1/2 activation. SP also induced association of the NK1R and src at the cell surface, determined by immunoprecipitation and western blotting. These Neuropeptides (2002) 36(5), 371–386

results suggest that b-arrestins are scaffolds for components of the MAP kinase pathway. Dominant negative b-arrestins, dynamin and rab5a inhibited resensitization of Ca2+ mobilization. Thus, receptor endocytosis and intracellular processing mediates resensitization. Many cells co-express several receptors for the same agonists. We evaluated the effects of activation of the NK1R on trafficking and signaling of the NK3R, and vice versa. Selective activation of the NK1R strongly inhibited endocytosis and homologous desensitization of the NK3R, but not vice versa. Over expression of b-arrestins reversed this inhibition. b-arrestins were required for endocytosis of both receptors. NK1R associated with both b-arrestin1 and 2 for prolonged periods in endosomes, which depleted cytosolic b-arrestins. In contrast, NK3R interacted with only b-arrestin2, and the association was transient. Thus, the NK1R sequesters b-arrestins in endosomes, thereby depleting cytosolic pools and preventing b-arrestin-dependent endocytosis and desensitization of the NK3R, which can remain at the cell surface and continue to signal. (Supported by NIH grant DK39957.)

Neuropeptide based drugs – did we blow it? John Hughes, University of Cambridge, UK. The excitement engendered in the early Ô70Õs by the discovery of the tachykinins, opioid peptides and numerous other neuropeptides was fuelled by the expectation that new therapeutic advances would arise from these discoveries. Attention was focussed on the development of receptor antagonists and in this task we and others were extremely successful. Unfortunately our expectations have been dampened by a series of negative clinical trials. I will review some of the early history of neuropeptide discovery and try to analyse where we may have erred and what we have learned in the development and clinical application of these discoveries. The premise that neuropeptide redundancy will inevitably blunt any antagonist effect will be examined.

Serendipity and the development of antagonists of substance P Alexander T. McKnight, Oxford Natural Products, Charlbury, Oxfordshire, UK. Of all the neuropeptides (save the opioids) substance P was seen as the most attractive of prospects for drug discovery. Even with role of substance P as a sensory transmitter ‘‘relegated’’ to that of modulator, the expectation was that an antagonist would be analgesic. The evidence from preclinical studies was strong (‘‘Target Validation’’), with knockout mice lacking the peptide or the NK1 receptor showing reduced responses to intense noxious, or inflammatory stimuli. When the first products of the drug-discovery process re-entered the preclinical arena, NK1 antagonists produced antinociceptive effects where there was inflammation (hyperalgesia), or nerve injury (hyperalgesia/allodynia). It was a surprise then, that the clinical studies with NK1 antagonists (‘‘Proof of Concept’’) against various types of pain (post-operative, postherpetic neuralgia, migraine) were negative. Explanations based on the marked species selectivity of NK1 antagonists were suggested, but the problem was not pharmacodynamic or pharmacokinetic because parallel studies showed positive effects using other end points. The rationale that arose for the clinical utilities of NK1 antagonists as anti-emetics or antidepressants was made empirically; although these discoveries were not accidental, they can reasonably be said to be serendipitous. It may be that the utility for NK1 antagonists in pain will be explored again when

Twelfth Annual Summer Neuropeptide Conference Abstracts

such compounds are established as novel therapies for other disorders of the nervous system.

Aminotetralin-derived neuropeptide Y Y5 receptor antagonists Mark A. Youngman1, James J. McNally1, Timothy W. Lovenberg2, Nicole M. Willard1, Allen B. Reitz1, Diane Nepomuceno2, Sandy Wilson2, Jerey Crooke1, Daniel Rosenthal1; Anil Vaidya1, Scott L. Dax*1, 1Johnson & Johnson, Pharmaceutical Research & Development, Welsh & McKean Roads, Spring House, PA 19477; and 23210 Merryeld Row, San Diego, CA 92121, USA. Starting from a lone micromolar screening lead, a diverse family of aminotetralin-derived neuropeptide Y Y5 antagonists has been developed. Refinement of Structure-Activity Relationships (SAR) and in vivo evaluation of select compounds in animal models of feeding/obesity will be presented.

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antagonist, des-Arg10Leu9 -kallidin (KD), inhibits hyperalgesia induced by noxious stimuli and B1 deficient mice have a diminished nociceptive response to such stimuli. Thus, a selective non-peptidic B1 receptor antagonist would represent a safe and novel approach to inflammatory pain therapies. PS309799 binds to human and macaque B1 receptors with Ki values of 3 and 30 nM, respectively. PS309799 is specific for B1 receptors vs. other GPCRs and is selective over the B2 subtype. Saturation binding experiments indicate that compounds bind in a competitive manner and are functional antagonists of the B1, but not the B2, receptor subtype. In the LPS-treated Green monkeys (Cercopithecus aethiops St-Kitts), the specific B1 agonist, des-Arg-KD, induced hypotensive and pro-inflammatory edema responses that were attenuated by PS309799. These data suggest that PS309799 and analogs are potent, selective, and efficacious B1 receptor antagonists.

Is there gold in that IP? Understanding the commercial value of biomedical research. Robert A. Bohrer, Professor of Law, California Western School of Law, San Diego, California.

Anorectic, anxiolytic, and antidepressant effects of a melaninconcentrating hormone1 receptor antagonist Christophe Gerald, Synaptic Pharmaceutical Corporation, Paramus, NJ, USA.

As biomedical research evolves with new tools, new discoveries, and new directions, patent law also evolves to provide new guidelines for intellectual property protection for the inventions produced by biomedical research. While researchers are now generally familiar with the fact that the gene sequences of many newly discovered genes have been patentable (and frequently patented) and that the discovery of new proteins may also be patentable, the requirements of utility and enablement required in order to patent those kinds of discoveries are continually changing. At the same time, as proteomics succeeds genomics as the overarching enterprise du jour, the role of particular proteins in disease processes, along with the use of those proteins in drug discovery has become an increasingly important research focus. The focus of this presentation will be on broad patent claims for the treatment of particular diseases by drugs that activate or inhibit a particular target. Some examples of such claims will be discussed, with special emphasis on the University of Rochester’s COX-2 (cyclooxgenase-2) patent, which was issued with the following claim:

The hypothalamic neuropeptide melanin-concentrating hormone (MCH) is important in the regulation of energy homeostasis and body weight. Although known MCH biological activity supports a rationale for the use of MCH antagonists in the treatment of obesity, it is not clear whether there is sufficient endogenous MCH tone to produce sustained loss of body weight after chronic MCH blockade. We evaluated SNAP 7941, a high affinity, selective MCH1 receptor antagonist in the diet-induced obesity model in rats. A robust and sustained decrease in food intake and body weight was observed which could not be attributed to conditioned taste aversion. Because MCH has also been implicated in the regulation of anxiety and mood, the compound was also assessed in a variety of animal models and species. SNAP 7941 reduced the number of vocalizations produced by guinea pig pups during a period of maternal separation in a manner comparable to the anxiolytic buspirone. In further support of its possible use as an anxiolytic, pretreatment with SNAP 7941 increased the degree of social behavior displayed by pairs of unfamiliar rats in the social interaction test. Similar to the profile of clinically used antidepressants, SNAP 7941 decreased immobility in the mouse forced swim test. These findings support the utility of an MCH1 antagonist for the management of obesity and its potential for the treatment of anxiety and/or depression.

The analysis of these broad ‘‘treatment’’ claims will focus particularly on the patentability requirements of utility and enablement. The identification of the receptors and pathways through which biologically active agents, including neuropeptides, operate has long been recognized for its scientific importance. As this presentation will emphasize, the value of that science, insofar as it enables the process of drug discovery and development, is also of enormous commercial importance.

Characterization of non-peptidic bradykinin B1 antagonists. M. Webb, M. Ohlmeyer, B. Strohl, V. Paradkar, D. Deblois*, Pharmacopeia, Princeton, NJ & U. of Montreal Hospital*, Montreal.

Technology and drug discovery: are we getting to the goal any faster? S. McLean, Dept. Neuroscience, Pfizer Global Research and Development, Groton, CT 06340, USA.

Bradykinin B1 receptors are expressed at negligible levels in normal tissues and are up-regulated following exposure to inflammatory cytokines and LPS. The selective peptidic B1

Technology holds the promise of delivering novel targets and the means of rapidly identifying small molecules that interact with these proteins. Successfully bringing a drug to market is a

1. A method for selectively inhibiting PGHS-2 activity in a human host, comprising administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product to a human host in need of such treatment.

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long, multistage process plagued by a high rate of failure. Thus, the need to identify technologies that facilitate discovery of novel biological targets and permit the rapid synthesis and screening of large numbers of compounds. The talk will focus on the discovery process and the integration of molecular approaches used to identify novel targets with high-speed chemical synthesis, molecular modeling, and rapid biological screening. Examples of screening for novel genes using microarray technology and strategies for identifying orphan receptors will be presented. Techniques for rapid followup of identified leads by high-speed chemistry and biology screening will also be discussed. Peptide biotechnology in Johnson & Johnson Allen B. Reitz, PhD, Welsh & McKean Rds, Johnson & Johnson Pharmaceutical Research Institute Spring House, Pennsylvania 19477. Johnson & Johnson is a major producer of recombinant protein products for therapeutic use, as demonstrated by the commercial success of ProcritÒ and RemicadeÒ. This talk will review how the world’s largest, broadly-based health care company incorporates biotechnology in many ways, including the application of newer molecular biology approaches to the discovery of medicines to treat debilitating human disorders.

Neuropeptides as drug targets for obesity Ildiko Antal, Bristol-Myers Squibb Pharmaceutical Research Institute, Department of Metabolic Research, POB 5400, Princeton, NJ 08543-5400, USA. Obesity is a major health problem in the Western Societies affecting a third of Americans. Even mild obesity enhances the risk of premature death, hypertension, diabetes mellitus, hyperlipidaemia, atherosclerosis, coronary heart disease, arthritis, sleep apnea, certain types of cancer and skin conditions and orthopedic problems. There is strong association of obesity and noninsulin dependent diabetes mellitus, more than 80% of NIDDM patients are obese. Therefore, one of the major challenges of the pharmaceutical industry and the healthcare system is to achieve sufficient, long-term weight loss to improve the overall health of the population. According to the Surgeon General obesity poses similar threat to the population as smoking. For an effective pharmacotherapy, understanding of the pathophysiology of energy homeostasis is essential. There are peripheral and central factors involved in energy homeostasis. The approaches to reducing body weight include: (1) Amplification of anorexigenic signals or blockade of orexigenic signals, (2) Blockade of nutrient absorption, (3) Increase thermogenesis, (4) Modulation of fat or protein metabolism and storage. This presentation will focus on the role of the central nervous system, especially neuropeptides and their receptors in maintaining energy balance. Particularly neuropeptides of the hypothalamus that seem to be important in the control of appetite will be discussed.

New concepts in the development and potential clinical application of somatostalin receptor-subtype selective peptides J. E. Taylor*, MD, Culler, Biomeasure Inc, Milford, MA, USA. Somatostatin (SS) was initially identified and characterized as an inhibitor of anterior pituitary growth hormone (GH) secretion. Subsequent studies have shown that SS exhibits multiple activities, including neural activity, antisecretory actions or pancreaNeuropeptides (2002) 36(5), 371–386

tic endocrine and exocrine functions, effects on gastrointestinal function, vasoactive activity, and antiproliferative actions in both normal and neoplastic tissues. With the introduction of cloning techniques, SS receptor genes encoding five subtypes (sst1–sst5) have been identified, and the modulation of SS activity by receptor-selective SS analogues may lead to novel and specific therapies for numerous disease states. Some examples include:  In addition to the known sst2-mediated antiproliferative actions on various tumors, there is also evidence that SS peptides may have a role in the treatment of benign proliferate diseases such as restenosis, atherosclerosis associated with organ transplantation, fibrosis, and undesirable angiogenesis-related conditions such as diabetic retinopathy. Both restenosis and atherosclerosis are the consequence of enhanced smooth muscle proliferation, whereas fibrosis results from excessive proliferation of the extracellular matrix. The SS receptor subtypes associated with these effects are not clearly known.  Vasoactive actions on hepatic-portal blood flow for the treatment of portal hypertension.  The potential clinical utility of selective ligands in the treatment of diabetic conditions. For example, recent studies have shown that sst5-selctive peptides more effective in inhibiting glucose-stimulated insulin secretion than the sst2 preferring peptides. This activity may lead to a treatment for the hyperinsulinemic state and other metabolic dysfunctions associated with the development of Type II diabetes.  The discovery and recent progress in the development of sst2–5 selective receptor peptides for pituitary adenomas. In addition to the well established utilization of sst2 selective SS agonists as inhibitors of pituitary GH secretion in the treatment of acromegaly, the application of SS receptor subtype pharmacology may lead to the potential treatment of other pituitary tumors such as prolactinomas or sst2 resistant tumors, with sst2–5 or sst5 selective SS analogues.

Neuropeptide Y (NPY) in regulation of blood pressure, cardiac contractility and other functions: lessons from NPY transgenic rats M. Michalkiewicz, Medical College of Wisconsin, Milwaukee. Given NPY’s colocalization with norepinephrine and other neurotransmitters together with the potent physiologic effects of exogenous peptide, it is important to determine the physiological functions of endogenous NPY. For this study we have developed NPY transgenic rats (NTR) in which NPY overexpression was targeted to its natural sites using a 14.5 kbp clone of the rat structural NPY gene. Relative to the nontransgenic littermates resting BP of NTR (but not locomotor activity) was significantly lower. Similarly during the stress conditions (novelty, forced swimming), BP was significantly lower in the NPY-Tg than in the control. NTR were not sensitive to an increased salt intake (8%) which increased pressure in control rats. Lower norepinephrine excretion in the NTR was associated with a reduced sympathetic tone to the heart and a lower basal and dobutamine-induced contractility of the left ventricle, while their longevity was significantly increased. These findings indicate that endogenous NPY may function as a powerful modulator acting to buffer excitatory adrenergic signaling. In the light of anticonvulsant and memory effects in these NTR such buffering action of NPY could be extended to glutamate- or GABAergic signaling. These findings indicate that genetic deficiencies in NPY or its receptors may be responsible for some forms of hypertensions, congestive heart failure and sodium-retaining disorders and that targeting NPY receptors with specific agonists or

Twelfth Annual Summer Neuropeptide Conference Abstracts

antagonists can offer new possibilities for treatment of these diseases. (Supported by NIH-HL57921.)

Polymorphisms of the corticotropin releasing hormone (CRH) promoter as genetic risk factor in rheumatoid arthritis C. G. Baerwald, M. Wahle, J. S. Lanchbury1, Med. Clinic and Policlinic IV, University Clinic, Leipzig, Germany; 1Molecular Immunogenetics and Rheumatology Units, United Medical and Dental Schools, Guy’s Hospital, London, United Kingdom. The regulatory region of the corticotropin releasing hormone (CRH) is highly conserved and plays a crucial role in the response of the organism to stress. Objective: Since it has been postulated that the impaired corticotropin releasing hormone (CRH) response to stress in patients with rheumatoid arthritis (RA) has a genetic basis, we investigated the distribution of CRH alleles in a cohort of UK patients as well as in South African RA patients. Methods: Restriction fragment length polymorphism of PCR amplified DNA products of the CRH promoter. We compared the allele frequencies in the RA patients with the respective healthy control population described previously. Results: As in the control populations we found two biallelic polymorphic sequences (named A1 and A2, and B1 and B2, respectively) in the CRH promoter which could be assigned to compound alleles. The A2B1 compound allele was protective against development of RA in a large group of UK Caucasoid patients (p = 0.03; odds ratio 0.43, 95% confidence interval 0.21– 0.88). In contrast, A1B1 was positively associated with RA in a cohort of black South African RA patients (p = 0.05; odds ratio 1.78, 95% confidence interval 1.01–3.15). Conclusion: Taken together, these findings support the hypothesis that CRH promoter polymorphism represents a new genetic marker for RA susceptibility and may prove useful for the prediction of RA risk in the future when further genetic and environmental risk factors are determined.

Familial diabetes insipidus and vasopressin polymorphisms Frederick D. Grant, Divisions of Endocrinology, Brigham and Women’s Hospital and Children’s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA, USA. Familial neurohypophysial diabetes insipidus (FDI) is an autosomal dominant disorder with typical onset in the first decade of life. Over thirty polymorphisms in the signal peptide and neurophysin regions of the vasopressin (Avp) gene have been associated with FDI, but the mechanism by which these polymorphisms cause vasopressin deficiency in heterozygous individuals has not been identified. Some autopsy studies have suggested loss of vasopressin-expressing neurons, but no animal model had existed for further study of FDI. Mice carrying a rat vasopressin transgene with a known mutation (G17V of neurophysin) develop a progressive polyuria with twice normal urine output by age 2 years. Development of complete DI may be limited by the short lifespan of the mouse. Histological analysis, including silver staining, has shown no evidence of neuronal degeneration. Cross-breeding of the FDI transgenic into vasopressin-deficient Avp)/) (knock-out) mice does not rescue the DI phenotype, suggesting impaired expression of the mutated gene from birth. Therefore, neuronal degeneration is not required for development of symptoms of FDI in mice and the FDI polymorphisms may cause a dominantly inherited VP deficiency by disrupting intracellular transport or processing of the VP peptide.

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Polymorphisms within neuronal genes correlating with predisposition to behavioural disorders act as neuronal specific differential regulators of gene expression’ J. P. Quinn*, A. MacKenzie1, V. J. Bubb, A. Scott, Physiological Laboratory & Dept Human Anatomy and Cell Biology, Crown St, University of Liverpool, Liverpool L69 3BX, [email protected] 1 Institute of Medical Sciences, Aberdeen AB25 2ZD. Genetic factors have been implicated in the aetiology of mental illness. Abnormalities in monoamine metabolism, in particular serotonin (5-HT) and dopamine have been implicated in the pathophysiology of many CNS related disorders. Clinical studies have suggested a polymorphism termed a variable number tandem repeat (VNTR) within intron 2 of the Serotonin transporter gene (5HTT) and the 3 untranslated region of the dopamine transporter (DAT1) gene are potentially associated with the susceptibility to such disorders. Although these clinical correlates have often been conflicting, the DAT 1 VNTR polymorphism has been more consistently correlated with attention-deficit hyperactivity disorder. Further the VNTR in the DAT1 gene can determine bioavailability of the transporters in vivo. Biochemically we have identified transcription factors binding the 5HTT VNTR by yeast one hybrid selection and demonstrated that a number of transcription factors will differentially regulate VNTR function. Previously clinical correlation with a specific disorder has been solely linked to copy number of the repeats, we have extended these studies further and demonstrated that individual repeat elements within the VNTR domain also differ in their enhancer activity in ES cells. This may in part explain the differential activity demonstrated by 5HTT VNTRs of different copy number (1). Thus clinical data should also address specific sequence of the VNTR in addition to copy number. We have demonstrated that these VNTR polymorphisms are functionally related in both binding similar transcription factors and in supporting tissue specific and differential enhancer activity. We have demonstrated that the 5HTT restricts marker gene expression in the developing CNS to areas associated with the initial endogenous 5-HTT expression (2). Similarly the DAT 1 gene strongly enhances transcription of a marker gene within DA neurones in organotypic adult rat CNS cultures (3). Our data clearly indicates a potential mechanism by which such regulatory polymorphisms might influence severity, onset or susceptibility to disease. They can act to vary the active complement of these crucial neurotransmitter regulators in a tissue specific and concentration dependent manner. These VNTRs are found in numerous other genes and potentially define a novel regulatory domain. (1) Fiskerstrand, C. E., Lovejoy, E. A., and Quinn, J. P. (1999) An intronic polymorphic domain often associated with susceptibility to affective disorders has allele dependent differential enhancer activity in embryonic stem cells. FEBS Lett 458, 171–174 (2) MacKenzie, A., and Quinn, J. (1999) A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo. Proc Natl Acad Sci USA 96, 15251–15255 (3) Michelhaugh, S. K., Fiskerstrand, C., Lovejoy, E., Bannon, M. J., and Quinn, J. P. (2001) The polymorphic domain termed a variable number of tandem repeats in the dopamine transporter gene functions as a transcriptional enhancer in dopamine neurons. Journal of Neurochemistry 79, 1033–1038. Neuropeptides (2002) 36(5), 371–386

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Induction of the dopaminergic phenotype in cultured retinal neurons by pituitary adenylate cyclase polypeptide PACAP J. M. C. Borba, M. S. G. Barreto, J. N. Hokoc¸ , F. G. deMello, IBCCF; CCS BI-G UFRJ, 21949-900, Brasil. In the present study, we examined whether the Pituitary Adenylate Cyclase Polypeptide (PACAP), a highly effective stimulator of adenylyl cyclase activity, might influence the differentiation of dopaminergic cells of the chick retina. Using cultured cells and tyrosine hydroxylase immunocytochemistry, we observed that tyrosine hydroxylase (TH)-positive cells were detected in cultures prepared from retinas at embryonic day 8, 9, and 10, after 6 days of culture. Cultures treated with 10 nM PACAP for 6 days showed a 2 to 3 fold increase in the number of TH–positive cells in cultures prepared from 9 and 10 dayold embryos. This effect was similar to that observed with culture treatment with forskolin (10 lM). Cultures treated with a specific antagonist of PAC-1 receptors (Maxd4; 500 nM), displayed a 50% reduction of the spontaneous differentiation of TH-positive cell, as compared to untreated cultures. Our results indicate a narrow window during development when undifferentiated dopaminergic cells may be influenced by specific signals conveyed by PACAP, possibly via the production of cAMP. Supported by CAPES; CNPq; FAPERJ and Pronex.

VIP Receptor antagonists reduce lung adenoma number in A/J mice James Dudek1, Halina Zakowicz1, Terry Moody1, Cloud Pawaletz2, Emmanuel Petricoin2, Robert Jensen3, 1Cell and Cancer Biology Branch, NCI, Rockville, MD, USA, 2Division of Cytokine Biology/CBER, PDA, Bethesda, MD, USA, 3Digestive Diseases Branch, NIDDK, Bethesda, MD, USA. The effects of VIP receptor antagoinsts on A/J mouse lung carcinogenesis were investigated. Lung adenomas developed in A/J mice 3 months after benz(a)pyrene or urethane injection. Three days after carcinogen injection, A/J mice were treated daily with VIPhyrid (VIPhyb; 10 lg, s.c.). VIPhyb, reduced lung adenoma number significantly by 42% using urethane as a carcinogen; similar results were obtained using benz(a)pyrene as a carcinogen. The protein expression profile in the mouse lungs was examined by surface enhanced laser desorption/ionization. The mouse lungs were examined for VIP receptors. By RT-PCR, VPAC1 but not VPAC2 receptor mRNA was detected in the mouse lung. Using immunocytochemistry, VPAC1 receptor immunoreactivity was detected in the lung adenomas, as well as the normal lung alveoli, bronchi and bronchioli. By receptor binding, specific 125I-VIP binding to mouse lung homogenates was inhibited with high affinity by (Lys15, Arg18, Leu27)VIP1– 7 GRF8–27, a VPAC1-R agonist, but not R025-1553, a VPAC2 agonist; both VIP and VIPhyb bind with high affinity to both VPAC1 and VPAC2 receptors. These results suggest that VIPhyb prevents lung carcinogenesis in A/J mice by blockade of lung VPAC1 receptors.

Activity-dependent neuroprotective protein (ADNP)-like immunoreactivity is localized to the nuclei as well as to the extracellular milieu of glial cells S. Furman1, R. A. Steingert1, J. Hauser1, D. E. Brenneman1, I. Gozes2, 1Clininal Biochem. Sackler Med. Sch., Tel Aviv Univ. Israel; 2SDMP, LDN, NICHD, National Institutes of Health, Bethesda, MD, USA. Neuropeptides (2002) 36(5), 371–386

ADNP is a glial-derived, vasoactive intestinal peptide (VIP)responsive protein. ADNP’s structure contains a nuclear export signal, 9 zinc fingers and a homeobox domain making it a potential transcription factor (J. Biol. Chem. 276, 708, 2001) NAPVSIPQ (NAP) is an 8 amino acid peptide derived from ADNP that showed protection of neurons from insults caused by several toxins. Specifically, NAP protected neurons from death caused by the beta amyloid peptide (J. Neurochem. 72, 1283, 1999). The concentration needed to protect neuron enriched cultures was higher than that needed to protect neurons in mixed neuroglial cultures (Regulatory Peptides 96, 39, 2000), suggesting the involvement of a glial derived factor. Taking together the ADNP structure motifs and the extracellular ADNP-derived peptide activity, we investigated subcellular localization and potential protein processing of the native protein in rat glial cultures. Antibodies were produced against the N terminal (anti-CNAP) and the C terminal (antiHGS) regions of ADNP. Cell fractionation, followed by western blot analysis localized intact ADNP-like immunoreactivity (IR, 120 kD) to the astrocytic nucleus as well as to the cytoplasm. Lower molecular weight bands exhibiting ADNP-like IR were observed, suggesting partial protein processing. Intact ADNPlike IR was also detected in the astrocyte-conditioned media and was increased following incubation with VIP. These results suggest that ADNP is a bi-functional protein that serves a nuclear role and may also be secreted from glial cells allowing interactions with other cells. Support: BSF, Neufeld, ISF, Gildor, ISOA, NIH.

The Nociceptin ORL1 system modulates anxiety like behavior in a modified social interaction test D. R. Gehlert1, C. Zink1, S. L. Gackenheimer1, S. D. Fitz2, A. Shekhar2, T. J. Sajdyk2, 1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA; and 2Indiana University Medical School, Department of Psychiatry, Indianapolis, IN 46202. The peptide Nociceptin was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain and produce behavioral effects in rodent models. Recently, nonpeptide agonists and antagonists of the ORL1 receptor have been reported in the literature. In the present study, we have evaluated the effects of Nociceptin as well as nonpeptide agonists and antagonists in a modified Social Interaction Test, a validated model of anxiety-like behavior. To study the effects of Nociceptin, bilateral cannulae were implanted into the basolateral nucleus of the amygdala (BLA) of Wistar rats. Nociceptin was infused into the BLA in a 100 nl volume of 1% BSA. The animals were assessed under high light, familiar conditions using the social interaction test with only the behavior of the treated animal scored. Nociceptin produced a dose-dependent increase in social interaction indicative of an anxiolytic-like effect. Subsequent studies were performed using i.p. administration of an analog of the nonpeptide agonist Ro 64-6198 and the ORL1 antagonist, J113397. Under high light, familiar conditions, the agonist produced an increase in social interaction. Under low light, familiar conditions, the antagonist produced a reduction in social interaction. These results are consistent with the proposed role of Nociceptin in anxiety-like behavior and suggest this system may be an interesting target for the discovery of novel anxiolytic agents.

Twelfth Annual Summer Neuropeptide Conference Abstracts

PAC1 expression and pacap hormone effects in Jurkat cells Patrizia Germano*, Mark Wu, D. J. Yamaguchi, Ken Tachiki, Joseph Pisegna, CURE/VA/UCLA, DDRC, West LA VA Medical Center, Los Angeles, CA 90073, USA. Introduction: PACAP has been indicated as an anti-inflammatory neuropeptide that, acting throughVPAC1 and VPAC2 receptors on T cells, is able to modulate their cytokine profile and to inhibit activation induced cell apoptosis (AICD): a very important mechanism for the manteinance of self-tolerance and for limiting lymphocyte proliferative responses to antigen stimulation. We have characterized for the first time the expression of native PAC1 receptors on a Jurkat T cell line and studied the effects of PACAP on tyrosine phosphorylation, cell proliferation and apoptosis. Methods: To demonstrate the expression of mRNA for native PAC1 receptors we have performed RT-PCR. To confirm the surface expression of PAC1 proteins, we have developed a fluorescent PACAP compound (Fluor-PACAP) and rabbit polyclonal antibodies directed against the COOH terminus of PAC1 and performed cytofluorometric and confocal microscopy analysis. Immunoprecipitation and Western blot analysis with anti-PAC1 rabbit polyclonal antibodies have been used to characterize PAC1 molecule. Jurkat cells have been activated by PACAP at 1, 3, 9, and 27 min and cells extracts and immunoprecipitates have been analyzed by Western blotting with anti-P-tyr mo-Abs. Cell proliferation and apoptosis were measured by MTT assay and cytofluorimetric analysis with Annexin V and propidium iodide. Results: Jurkat cells expressed native PAC1 at mRNA level and PAC1 receptors on their membranes. Immunoprecipitated PAC1 receptor showed a MW of 50 Kd. A net inhibition of tyrosine phosphorylated bands was detected between 3–27 minutes, upon PACAP activation, by Western blotting. Jurkat cells incubated with serial dilutions of PACAP, in the presence or absence of immobilized anti-CD3 mo-Abs, presented a rate of proliferation that appeared increased by anti-CD3 and PACAP, and decreased by PACAP only, in a dose-response manner. An increase of apoptosis, following PACAP stimulation, was confirmed by cytofluorimetric analysis with Annexin V-FITC and propidium iodide staining. FasL expression appeared to be down-regulated by both PACAP and anti-CD3 activation. Conclusions: We showed for the first time the membrane expression of high affinity PACAP receptors on human leukemic T cells. PACAP activation appeared to protect Jurkat cells from antiCD3 induced apoptosis, whereas PACAP was able to inhibit a tyrosine signaling cascade and to increase the apoptotic index when added by itself. Further studies are necessary to explore a possible application for PACAP to modulate tumor T cell growth. Neurokinin B-mediated plasma extravasation in tachykinin NK1 receptor wild-type and knockout mice Andrew D. Grant, Roksana Akhtar, Susan D. Brain, Cardiovascular Biology and Medicine, King’s College, London, UK. Neurokinin B (NKB) has been implicated in the hypertension which characterises pre-eclampsia, a condition where tissue oedema formation is common. The ability of NKB (i.d or i.v) to induce oedema formation was examined by accumulation of 125 I-BSA in NK1 receptor wild-type (+/+) and knockout ()/)) mice. I.d NKB caused a doss-dependent increase in plasma extravasation over 30 min in +/+ mice (p < 0.05), with no effect on )/) mice. I.v NKB to +/+ mice produced plasma extravasation in skin, uterus, liver and particularly in the lung of the mice (p < 0.01), and this was not mirrored by substance P. Furthermore, in NK1 receptor )/) mice, NKB only led to

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plasma extravasation in the lung and liver. The plasma extravasation produced by NKB in the lungs of )/) mice was unaffected by treatment will the NK2 and NK3 antagonists SR4 8968 and SR142801. We conclude that NKB is a potent stimulator of plasma extravasation through two distinct pathways. One is via the wellcharacterised activation of NK1 receptors, and the other appears to be a novel neurokinin receptor-independent pathway specific to NKB. Endogenous neurotensin (NT) modulates nociceptive and stress induced anti nociceptive responses to noxious visceral stimulation: demonstration in NT antagonized rats and in NT knockout mice Xianyong Gui, Paid Dobner, Robert E Carraway, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. Neurotensin (NT) exerts bipolar effects on pain perception, facilitating at low doses (pM) and inhibiting at high doses (nM) when given centrally to rodents. The NT system is upregulated by stress and NT is implicated as a mediator of stress-induced anti-nociception (SIAN). We studied visceral pain sensitivity (VPS) by measuring visceromotor responses (abdominal muscular contraction) to colorectal distension under non-stress and post-stress conditions (30 min-cold water avoidance) in rats given NT-antagonist SR48692 (0.5mg/kg) and in NT-knockout mice as compared to controls. SIAN was observed in control rats and mice (n = 6), where VPS was significantly lower (p < 0.05) after stress than in the non-stress condition. In contrast, SIAN was not observed in rats given SR48692 and in NT-knockout mice, where VPS was higher after stress than in the non-stress condition. SR48692 had little effect on VPS in non-stressed rats, but it enhanced VPS in stressed rats. In NTknockout mice, VPS was severely depressed as compared to wild-type control, except in the post-stress condition. These data, demonstrating that VPS can be altered by acute as well as chronic withdrawl of NT, suggest important role(s) for NT as a regulator of nociception and modulator of SIAN. PAC1 and PACAP expression, signaling, and role in growth of HCT8 human colonic tumor cells Sang V. Le, Patrizia M. Germano, Mark Wu, Dean J. Yamaguchi, Joseph R. Pisegna*, CURE: Digestive Diseases Research Center. VA Greater Los Angeles Healthcare System and Dept of Medicine. University of California. Los Angeles. Los Angeles, California 90073, USA, *Corresponding author: UCLA/CURE: DDRC, VA GLAHS. Bldg 115, Room 203, 11301 Wilshire Blvd. Los Angeles, CA 90073 Tel.: +310-478-3711 ext. 41940; fax: +310-268-4096; E-mail address: [email protected] (J. R. Pisegna). The PACAP type 1 receptor (PAC1) is a hepta-helica1, G protein coupled receptor that has been shown to be expressed on nonsquamous cell lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in native colonic tissue. Polyclonal antibody directed against the COOH terminal of PAC1 was used to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells as well as with Fluor-PACAP using PACS analysis and confocal laser scanning microscopy. RT-PCR using specific PAC1 primers confirmed expression of PAC1 on HCT8 cells. In HCT8 cells, PACAP-38 elevates cAMP and intracellular protein phosphorylation in a dose-dependent manner with a half-maximal (EC50) Stimulation of approximately 3 nM. Neuropeptides (2002) 36(5), 371–386

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PACAP-38 dose dependently stimulates proliferation as shown using the MTT assay. In addition, HCT8 colonic tumor cells expresses the cell surface receptor Fas, which is down regulated upon activation with PACAP-38. These data indicate that HCT8 human colon cancer cells express PAC1, release PACAP hormone and stimulate adenylyl cyclase and cellular proliferation. Therefore, PACAP is capable of increasing the growth of a human colonic cancer cell line suggesting that PACAP might play a role in the regulation of colon cancer growth and modulate T-lymphocytc responses against tumor cells.

Primary phenotypic characterization of mice with a targeted vip gene disruption: comparison with PACAP KO mice. Vincent Lelie`vre, Christopher Colwell, Zhongting Hu, Yevgenyia loffe, Tannaz Razinia, James A. Waschek, Department of Psychiatry and Mental Retardation Research Center, Neuropsychiatric Institute, University of California at Los Angeles, Los Angeles, CA, USA. Vasoactive intestinal peptide (VIP) belongs to a family of peptides hat includes PACAP, secretin and glucagon. Because VIP and PACAP share common receptors called VPAC1 and VPAC2, they share some of the biological actions. These neuropeptides have been reported to act in the process of neurotransmission, and in the modulation of cell growth and tissue development. Using gene disruption approaches in mice, we have begun to examine the common and independent in vivo functions for VIP and PACAP. We have generated VIP altered mice by inserting a neomycin cassette within the exon 4 of the VIP gene. PCR analysis, RIA assays and immunohistochemistry have been performed to show lack, or near lack, of VIP production in animals homozygous for the disrupted gene. These mice are viable and fertile. In parallel, we also generate mice PACAP knockout (KO) mice. Initial phenotypic comparisons have been made between these mice and wild type controls. Using SHIRPA protocols, preliminary data showed that VIP and PACAP animals both show alterations in the locomotor activity and several other sensorimotor abnormalities. Mice with mutations in the VIP and PACAP genes also show pronounced, but distinct abnormalities of circadian rhythms. Reduced norepinephrine release in transgenic rats overexpressing neuropeptide Y. T. Michalkiewicz, S. J. McDougall, M. Michalkiewicz, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee. Neuropeptide Y (NPY) is expressed in catecholaminergic neurons of the central and of the peripheral sympathetic nervous systems. We have tested a hypothesis that in long-term regulation endogenous NPY acts to reduce norepinephrine (NE) release and to decrease sympathetic nervous system activity. For this study we have generated transgenic Sprague Dawley rats (NTR) overexpressing NPY under its natural promoter. NPY overexpression was detected in a number of transgenic tissues. NE concentrations were measured (radioenzymatic assay, Amersham) in urine collected over 24-hr period from undisturbed male rats. Comparing to non-transgenic littermates, urine levels of NE were significantly (p < 0.05) reduced in NTR. Blood samples for plasma levels of NE were collected from conscious rats via chronic arterial catheters. Both strains had a similar level of circulating NE at the baseline session but they differ during the stress. Accordingly, a highly significant increase in plasma NE was observed in nontransgenic littermates but not in the NTR. A significant inhibitory effect of the genotype was found on this Neuropeptides (2002) 36(5), 371–386

measure (P = 0.038). This finding indicates that NPY upregulation is associated with a reduced activity of the sympathetic nervous system in baseline and with a diminished an immediate adrenergic response to a stress. Thus, endogenous NPY may function in the sympathetic rervous system as an inhibitor of adrenergic signaling. Such buffering action of endogenous NPY may have a neuroprotective effect important in hyperexcitability states. (Supported by: NIH HL57921.)

The role of vasopressin in anxietyrelated behavior in rats Helen M. Murphy, Cyrilla H. Wideman, John Carroll University, Cleveland, OH 44118, USA. The neuropeptide, arginine vasopressin (AVP), was studied in association with anxiety, as measured by the elevated-plus maze. Vasopressin-containing, Long-Evans (LE) and vasopressin-deficient (DI) rats were placed on the maze for a 5-minute time period. Total time and entries on open arm, closed arm, and center square were recorded. For open arm entries, time was significant, strain was not significant, and there was an interaction between time and strain over the 5-minute period. Overall, the LE animals entered the open arm of the maze significantly more than the DI animals. However, during the first minute, the DI animals entered the open arm significantly more than the LE animals. Results suggest that AVP plays a role in anxiety-related behavior.

Chromogranin-A (CGA) and synaptophysin as markers in assessing the neuroendocrine component in colorectal cancer David Oh, Peter Park, Jerome Wollman, Klaus Lewin, Joseph Pisegna*, VA Greater Los Angeles Healthcare System and Department of Medicine, University of California Los Angeles, Los Angeles, CA, USA. Purpose: Adenocarcinoma of the colon and rectum is a significant health problem; however, no previous study has identified whether neuroendocrine cells are a tumor-promoting component of colorectal cancers or whether their expression reflects more aggressive tumor growth. Neuroendocrine cells contain bioactive amines and peptides, which may have an Important role in promoting the growth of transformed cells such as in adenocarcinomas. Accordingly, this retrospective study was designed to determine the frequency by which markers of neuroendocrine cells are expressed in colorectal cancers. Methods: 15 randomly selected paraffin-embedded tissue sections were obtained from archival tissues from 1998 to 1999, which varied in their stage and grade. Immunohistochemistry was performed, following de-paraffinization using specific stains for both CGA and synaptophysin. The pattern of staining was graded on a scale and correlated with negative and positive controls. H&E staining correlated the results with grading of tumors. A single pathologist (KL) who was blinded to the tumor grade and extent of disease reviewed all slides. Results: CGA was expressed in 10/15 (67%) of colorectal cancers, 5/15 (33%) showed weak positive staining and 5/15 (33%) with strong positive staining, 5/15 (33%) of the tumors showed no CGA staining. Tumor cells expressing synaptophysin were identified in 12/15 (80%) of cases; 27% with weak positive staining while 53% of cases showed strong positively, 4/15 (27%) of tumors showed strong positive staining for both Chromogranin-A and synaptophysin. On retrospective analysis, all four of these tumors showed advanced Dukes C staging and metastatic spread. Conclusions: CGA and synaptophysin are positive in a percen-

Twelfth Annual Summer Neuropeptide Conference Abstracts

tage or advanced colorectal adenocarcinomas. Synaptophysin has a greater positive predictive value compared to CGA. Strong positive staining with both CGA and Synaptophysin correlated with more aggressive grades of adenocarcinomas. These results indicate that an expansion of neuroendocrine cells in colorectal adenocarcinoma may be involved in promoting tumor growth as detected with specific stains and may have significant clinical implications such as for prognosis of disease and deserves further studies. Substance P–immunoreactive neurons in the central nervous system of the pulmonate gastropod Laevicaulis alte (FERUSSAC) L. Patnaik, M. Pani, S. Das, D. R. Naik, Dept. of Zoology, Utkal University, Bhubaneswar, 751004, India. Immunohistochemical methods have helped the detection of a number of signal peptides in the molluscan nervous system. Although substance P merits importance for its participation in neural communication and/or involvement in neuropharmacological impositions, it has not received adequate attention in molluscs. Using me PAP immunohistochemistry, we studied the distribution of SP-ir neurons in the central nervous system of the terrestrial pulmonate, Laevicaulis alte. The immunoreactive cell bodies were localised in the cell rind of all the central ganglia in L. alte. In the cerebral ganglia SP+ neurons were present in the postcerebrum, whereas they were absent in the pro- and mesocerebrum. The SP labeling in the postcerebrum was restricted to a few oval or elongated cells measuring 15–50 lm in diameter (small to medium size). They occurred as solitary cells in the ventrolateral margin of the cerebral ganglia and showed dense immunoreactivity. In comparison to the cerebral ganglia, the pleural ganglia had more number of cells but they showed relatively weak immunoreactivity. The SP+ neurons in the pleural ganglia were of more or less uniform size; the neuronal perikarya were round or oval in shape with a central nucleus. A few solitary SP+ neurons were observed in the outer margin of the parietal ganglia These were medium-size neurons, oval or elongated in shape with a round or oval central nucleus and showed a dense immunoreactive cytoplasm. In the pedal ganglia the labeled neurons occurred in clusters along the dorsolateral margin. They were also of medium size, round shape and contained dense immunoreactive cytoplasm. Solitary SP+ neurons were detected in the an anterodorsal margin of the visceral ganglion. These were small to medium-size neurons with an elongated perikarya and a central oval nucleus. Besides the ganglia, interestingly, a few small and weakly immunoreactive SP+ neurons were observed in the cerebral commissure along its ventral margin, close to the postcerebrum. SP-ir fibers with occasional varicosities were also observed in the cores of the cerebral, pleural, pedal and visceral ganglia. These ganglia are involved in a variety of physiological and peripheral functions in gastropods. Although the exact function of substance P in gastropods remains elusive, this immunohistochemical data implies profound significance of SP in gastropods. It may also suggest a diversity in its central and peripheral functions. Free radical release from activated microglial cells is attenuated by the neuregulin glial growth factor2 (GGF2) F. O. Dimayuga1, Q. Ding2, J. N. Keller1,2, M. A. Marchionni3, K. B. Seroogy1, A. J. Bruce-Keller1, 1Department of Anatomy & Neurobiology, 2Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536; and 3CeNeS Pharmaceuticals, Inc. Norwood, MA 02062, USA.

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The neuregulin glial growth factor 2 (GGF2) is a polypeptide growth/differentiation factor that is perhaps best known for it’s ability to promote the survival and proliferation of oligodendrocytes and Schwann cells. While it has been shown in recent years that GGF2 is effective in the treatment of autoimmune models of brain injury, it is not known if the beneficial effects of GGF2 are based in part on modulation of brain inflammation. In this study, we document the anti-inflammatory effects of recombinant human GGF2 (rhGGF2) on microglial free radical production in vitro. The presence of the neuregulin tyrosine kinase receptors ErbB2, 3, and 4 was confirmed in murine N9 microglial cells by Western blot analysis. Pretreatment of N9 cells with rhGGF2 24 hours before either PMA or IFNc caused dose-dependent decreases in oxidative burst activity and nitrite release, respectively. When cells were co-treated with increasing doses of rhGGF2 and PMA or IFNc, only concentrations of 50 ng/ml, but not 10 or 100 ng/ml, were able to decrease oxidative burst activity and nitrite release. Finally, when microglial cell viability following treatment of cells with IFNc, with or without rhGGF2, was evaluated, it was found that rhGGF2 dose-dependently conferred significant protection against IFNc-induced cell death in the microglial cells. Overall, these results indicate that the neuregulin GGF2 may have antiinflammatory and anti-oxidant properties in the brain, and may also provide trophic support for brain-resident microgical cells.

Neuropeptides affect cytokine and chemokine production in dendritic cells Evros Vassiliou, Mario Delgado, Xiuju Jiang, Doina Ganea, Rutgers University, Newark, NJ, USA. Dendritic cells (DCs) are major participants in both innate and adaptive immunity. Exposure to pathogens results in DC maturation, i.e. changes in phenotype, cytokine and chemokine expression, and migration to lymphoid organs. Although neuropeptides are present at relatively high levels in areas rich in DCs, the role of neuropeptides in DC maturation and function is largely unknown. We reported previously that vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as anti-inflammatory agents by deactivating macrophages. Here we determined their effects on myeloid DCs. Enriched DCs (70–80% CD11c+), as well as purified DCs (>90% CD11c+) were stimulated with LPS, PGN, or antiCD40 Abs, in the presence or absence of V1P or PACAP. DCs constitutively express all three types of VIP/PACAP receptors, VPAC1, VPAC2, and PAC1, and the neuropeptides inhibit both TNFa and bioactive IL-12 production in a dose-dependent manner. In addition to proinflammatory cytokines, DCs are major producers of chemokines. Two of these chemokines affect Th1/Th2 migration in opposite ways. IP-10 (CXCL10) acts on the CXCR3 receptor expressed by Th1 effectors. In contrast, MDC (CCL22) acts on the CCR4 receptor expressed by Th2 cells. VIP and PACAP inhibit IP-10, and enhance MDC production in LPSstimulated DCs, and this effect correlates with the chemoattraction of Th2, but not Th1 cells, by neuropeptide-pretreated DCs. These results are in agreement with previous reports that VIP and PACAP favor in vivo Th2-type responses, which represents another angle of their anti-inflammatory activity.

High dose CRF pretreatment increases subsequent CRF metabolism in rat pituitary cultures J.C. Ritchie*, C. Ramsey, Emory University Sch. of Med., Atlanta, GA 30322. Neuropeptides (2002) 36(5), 371–386

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To evaluate the importance of post-translational processing in the regulation of Corticotropin Releasing Factor (CRF) we studied the metabolism of this peptide in acute rat pituitary cultures. Cultures were established from male Sprague Dawley rats. Pituitaries were enzymatically dissociated and planted in 48 and 24 well plates (Vale et al., Methods in Enzymology, 103, 1983). For these experiments, cultures received either: no treatment, CRF vehicle (0.1 mM acetic acid), 25 nM CRF, or 50 nM CRF. Treatments were repeated twice at three hour intervals. Two hours after the last treatment, media were removed and the cells wers washed twice with fresh media. The cultures were incubated one hour, the media removed, and fresh media applied. A mixture of iodinated and uniodinated r/h CRF (1.25

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nM) was then added. After three hours, media and cells were harvested. Media was assayed for ACTH and chromatographed (HPLC) to assess CRF metabolism by UV absorbance and radioactivity recovery. A second series of experiments incorporated the use of concanavalin A, PAO, and R121919 ( CRF antagonist) to test the effects of blocking CRF endocytosis. In general, cultures initially exposed to high concentrations of CRF, released less ACTH into their media than those with no previous exposure. Additionally, iodinated-CRF peaks were smaller in pretreated cultures. CRF metabolite peaks tended to be larger in those cultures receiving pretreatment but this finding did not reach statistical significance. (Supported by NIMH grant MH59833.)