Neuropeptides 2004

Neuropeptides Neuropeptides 38 (2004) 385–424 www.elsevier.com/locate/npep

Abstracts

NEUROPEPTIDES 2004 XIV European Neuropeptides Club Meeting Alicante, 9–12 May 2004 Neuropeptides 2004 Organization Chair Carlos Belmonte, Spain

Scientific Programme Committee Adolfo Aracil (Secretary), Spain Susan Brain, UK Juana Gallar, Spain Pierangelo Geppetti, Italy Robert F. Schmidt, Germany

European Neuropeptides Club Chairing Committee Daniel Hoyer, Chair, Switzerland Manfred Zimmermann, Vice-chair, Germany Illana Gozes, Secretary, Israel Riccardo Patachini, Treasurer, Italy Carlos Belmonte, Meeting Adviser, Spain

Programme Overview

Sunday, 9th May 2004 Evening: 18:00 18:30

Welcome address, official opening of Neuropeptides 2004 ‘‘Manfed Zimmermann Award’’ to Professor Janos Szolcsa´nyi (Hungary) Special Plenary Lecture: Forty years in capsaicin research for sensory pharmacology and physiology Chair: Carlos Belmonte; Laudation: Manfred Zimmerman.

Monday, 10th May 2004 Morning: 9:00–10:00

10:15–12:00.

Keynote Lecture 1: Sensory neuropeptides and addiction Carmen De Felipe (Spain) Chair: Robert F. Schmidt Meeting Symposium 1: Calcitonin gene-related peptide (CGRP) involved in meningeal nociception and headache Chair: Karl Messlinger (Germany) 1. Karl Messlinger (Germany) Role of neuropeptides in primary headaches – current concepts

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2004 Published by Elsevier Ltd. doi:10.1016/j.npep.2004.09.002

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12:00–13:00

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2. Susanne Sauer (Germany) CGRP release from skin, nerve and dura mater in vitro 3. Richard Carr (Germany/Australia) Neurobiology of CGRP release in the dura mater 4. Katharina Zimmermann (Germany) Sensitising effects of CGRP in nociceptive afferents 5. Michael Fischer (Germany) BIBN4096 reduces spontaneous activity of neurons with meningeal input 6. Kirsten Arndt (Germany) Preclinical and clinical characterization of the first non-peptide CGRP receptor antagonist, BIBN 4096BS Poster Session 1.

Afternoon & Evening: 16:00–17:00 Keynote Lecture 2: Pathophysiological role of urotensin II in mammals. Riccardo Patacchini (Italy) Supported by Menarini Ricerche Chair: Manfred Zimmermann 17:15–19:00 Meeting Symposium 2: Nociceptin/orphanin FQ and its receptors-novel ligands and therapeutic opportunities Chair: Girolamo Calo` (Italy) 1. Jean-Claude Meunier (France) How does the nociceptin/orphanin FQ receptor bind peptides? 2. John McDonald (UK) Novel potent peptide ligands for the nociceptin/orphanin FQ receptor 3. Girolamo Calo´ (Italy) Blocking Nociceptin/orphanin FQ signalling produces antidepressant like effects in rodents 4. Niall P. Murphy (Japan) Nociceptin/orphanin FQ control of reward: pharmacological and genetic studies 5. Michele Morari (Italy) Nociceptin/orphanin FQ receptor antagonists reverse akinesia in parkinsonism models 19:45 Official welcome reception hosted by the Alicante Municipality at the Santa Barbara Castle.

Tuesday, 11th May 2004 Morning: 9:00–10:00

10:15–12:00.

Keynote Lecture 3: Neuropeptides in control of sleep and wakefulness Luis de Lecea (USA) Chair: Robert F. Schmidt Meeting Symposium 3: Neuropeptides in immunity Co-chairs: Illana Gozes (Israel) and Mario Delgado (Spain) 1. Eberhard Weihe (Germany) Neuropeptide signatures in immune functions and disfunctions 2. Mario Delgado (Spain) VIP: a ‘‘very important peptide’’ in immunomodulation. Potential therapeutic implications 3. Rosa P. Goma´riz (Spain) VIP decreases the expression of Toll-like receptors 2 and 4 in a mice model of Crohn’s disease

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12:00–13:00.

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4. James Washeck (USA) PACAP and VIP: reciprocal immune/neural modulators after nerve injury 5. Javier Leceta (Spain) VIP induces tolerogenic differentiation of immune cells 6. John Quinn (UK) Expression and function of substance P in non-neuronal cells correlated with infection and immunity 7. Leo Hofland (The Netherlands) Somatostatin and cortistatin in the human immune system. Poster Session 2.

Wednesday, 12th May 2004 Morning: 9:00–10:30

10:45–12:45

12:45

Meeting Symposium 4: Hot topics and last advances Co-chairs: Daniel Hoyer (Switzerland) and John Taylor (USA) Supported by IPSEN and Novartis 1. John Taylor (USA) Novel aspects of the Ghrelin analoges: the dissociation of GH secretion from weight gain 2. Daniel Hoyer (Switzerland) Somatostatin receptor agonists/antagonists in CNS disease models 3. Illana Gozes (Israel) From VIP and PACAP to ADNP and NAP: new horizons in brain development and neuroprotection 4. John Quinn (UK) Regulation and Function of the Tachykinins 5. Jerzy Nowak (Poland) Functional characteristics of peptide histidine-isoleucine (PHI) and peptide histidine-methinine (PHM) versus VIP and PACAP in vertebrate brain Meeting Symposium 5: Selected communications Co-chairs: Adolfo Aracil and Juana Gallar General Assembly, Concluding Remarks of Neuropeptides 2004, and introduction of Neuropeptides 2005.

Keynote Lectures Sunday, May 9th 18:30 h ‘‘Manfred Zimmermann Award’’ Special Plenary Lecture Forty years in capsaicin research for sensory pharmacology and physiology Professor Janos Szolcsa´nyi Monday, May 10th 9:00 h Keynote Lecture 1 Sensory neuropeptides and addiction Carmen de Felipe Monday, May 10th 16:00 h Keynote Lecture 2 Pathophysiological role of urotensin II in mammals. Riccardo Patacchini Supported by Menarini Ricerche Tuesday, May 11th 9:00 h Keynote Lecture 3 Neuropeptides in control of sleep and wakefulness. Luis de Lecea

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Forty years in capsaicin research for sensory pharmacology and physiology J. Szolcsa´nyi, Department of Pharmacology, University Medical School of Pe´cs, Neuropharmacology Research Group of the Hungarian Academy of Sciences, Pe´cs, Hungary Pharmacology of the peripheral nervous system is highly one sided. Broad spectrum of drugs which modulate efferent neurotransmissions are available for patients while drugs acting selectively on its afferent side are almost completely missing. I have been convinced since the late sixties that capsaicin is a powerful lead molecule which is suitable for opening the magic box of sensory pharmacology. Structure-activity relationships, psychophysical assessments in ourselves and the remarkable lack of its irritancy in birds and frogs were the cornerstones for postulating the existence of a capsaicin receptor (Szolcsa´nyi and Jancso´-Ga´bor, 1975). Electrophysiological recording from whole sensory nerves or single units provided the first evidence for selective excitation and desensitization of C-polymodal nociceptors (Szolcsa´nyi, 1977, 1980). Furthermore, thermoregulatory behaviour of capsaicin-treated rats suggested also an action on peripheral and hypothalamic warmth sensors (Jancso´-Ga´bor et al., 1970a,b; Szolcsa´nyi et al., 1971). Pharmacology of capsaicin finally led to molecular identification and cloning its VR1 or TRPV1 receptor (Caterina et al., 1997) and in fact the whole family of TRPV cation channels. Furthermore, since the TRPV1 cation channel is gated by noxious chemical stimuli it turned out to be a promising target for initiation high throughput screening for drug development of analgesics. Capsaicin has opened new scopes also in the field of sensory physiology and pathophysiology. Utilising its neuroselective actions direct evidence was obtained for neurogenic inflammation (Jancso´ et al., 1967) and new types of neurogenic smooth muscle responses mediated by capsaicin-sensitive sensory nerves were discovered (Szolcsa´nyi and Bartho´ 1978,1982). The concept of dual ‘‘sensory-efferent’’ function of these ‘‘chemoceptive’’) nociceptors was formulated (Szolcsa´nyi, 1984). In further studies the mediator background (tachykinins, CGRP) and large scale of tissue responses mediated by capsaicin-sensitive nerves were described (Holzer, 1992; Maggi, 1995). New chapter has been opened when it turned out that somatostatin released from these nociceptors reaches the circulation and elicits systemic ‘‘sensocrine’’ antiinflammatory and antinociceptive effects (Szolcsa´nyi et al., 1998a,b).

Sensory neuropeptides and addiction C. De Felipe a, P. Murtra a, C.A. Gadd b, S.P. Hunt b, a Instituto de Neurociencias, Universitas Miguel

Herna´ndez, Ap. Correos 18, San Juan de Alicante 03550, Spain, b Department of Anatomy and Developmental Biology, University College London, London, WC1E 6BT, UK E-mail: [email protected] Modulation of substance P activity offers a radical new approach to the management of depression, anxiety and stress. The substance P receptor is highly expressed in areas of the brain that are implicated in these behaviours, but also in other areas such as the nucleus accumbens which mediate the motivational properties of both natural rewards such as food and of drugs of abuse such as opiates. We show a loss of the rewarding properties of morphine and other drugs of abuse in mice with a genetic disruption of the substance P receptor. The loss was specific to morphine, amphetamine, alcohol and nicotine, as both groups of mice responded when cocaine or food were used as rewards. The physical response to opiate withdrawal was also reduced in substance P receptor knockout mice. We conclude that substance P has an important and specific role in mediating the motivational aspects of opiates and may represent a new pharmacological route for the control of drug abuse. To identify the areas of the brain that might contribute to this effect, we assessed the behavioural effects of ablation of neurons expressing the NK1 receptor in specific regions of the mouse brain using the neurotoxin substance P-saporin. Loss of NK1 receptor-expressing neurons in the amygdala caused an increase in anxiety-like behaviour and also a reduction in morphine CPP scores, without affecting CPP to cocaine. NK1 receptor-expressing neurons in the mouse amygdala therefore modulate morphine reward behaviours. These observations mirror those observed in NK1 receptor knock-out (NK1)/)) mice and suggest that the amygdala is an important area for the effects of SP and the NK1 receptor in the motivational properties of opiates, as well as the control of behaviours related to anxiety.

Pathophysiological role of urotensin II in mammals R. Patacchini a, P. Santicioli a, P. Rovero b, S. Giuliani a, C.A. Maggi a, a Department of Pharmacology, Menarini Ricerche, Florence, Italy, b Department of Pharmaceuticals Sciences, University of Florence, Florence, Italy E-mail: [email protected] Urotensin II (U-II) is a cyclic dodecapeptide originally isolated from the teleost fish neurosecretory system, and subsequently identified in other species, including man. The interest on human U-II (hU-II)-a undecapeptide retaining the cyclohexapeptide sequence

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of fish U-II-has grown enormously in the last few years, following the identification of a specific human receptor (formerly GPR14/SENR orphan receptor), termed UT receptor (Ames et al., 1999). Various studies aiming at investigating the pathophysiological role played by this peptide in mammals have shown that hU-II produces a very potent vasoconstriction of cynomolgus monkey arteries and human arteries and veins, and potently contracts certain blood vessels from other species. hU-II has been found 1–2 orders of magnitude more potent than endothelin-1 in producing vasoconstriction, so that it has been proposed to be the most potent mammalian spasmogen identified. However, the vasoactive responses to hU-II undergo large variations, between and within species, as for the type of vessels sensitive to this peptide than for the contractile efficacy shown by hU-II. hU-II also produces in vitro and in vivo vasodilation of certain vascular beds, and exerts potent inotropic effects in the human heart in vitro. On the basis of its spectrum of activities, hUII has been suggested to modulate cardiovascular homeostasis and possibly to be involved in certain cardiovascular pathologies as atherosclerosis, hypertension, cardiac hypertrophy, renal dysfunction and diabetes. Both U-II and its UT receptor have been detected in the mammalian brain and spinal cord, implying a possible neuromodulatory role of this peptide in the CNS. Novel UT receptor antagonists have been developed possessing increased affinity and selectivity for the UT receptor over the first compounds, which should help to clarify the pathophysiological role played by this peptide.

Reference Ames, R.S., Sarau, H.M., Chambers, J.K., et al., 1999. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401, 282–286.

Neuropeptides in control of sleep and wakefulness L. de Lecea, Departments of Molecular Biology and Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037, USA E-mail: [email protected] Aim of investigation. Neuropeptides have long been considered as orchestrators of the main neurotransmitter systems that modulate the sleep/wake cycle. In particular we have focused our research on two neuropeptides that have a key role in the regulation of the states of vigilance: hypocretins and cortistatin. The hypocretins have been associated with the narcolepsy, a sleep disorder characterized by intrusions of REM sleep into wakefulness. Therefore the hypocretins play a key role in the stability of arousal networks. The interaction

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between the hypocretins and the monoaminergic systems and the role of the hypocretins in mediating the hyperaroused state associated with stress will be discussed. Cortistatin is another neuropeptide with an important role in stabilizing cortical circuits during sleep. We have generated mice that lack cortistatin and analyzed sleep and wakefulness in these mice. Results. Injection of hypocretin-1 (10 pmol) into the locus coereuleus region of rats significantly increased wakefulness and suppressed REM sleep during the 4 h of sleep recordings. Intracerebroventricular administration of cortistatin in rats and mice causes dramatic changes in sleep architecture. Mice deficient in cortistatin show an increase of sleep episodes in the dark cycle, which suggests a role for cortistatin in the stability of cortical synchronization during sleep. Conclusion. Peptidergic systems are important regulators of the neurotransmitter networks that dictate the states of vigilance. Acknowledgement. This work was supported by grants from NIH.

Role of CGRP in nociception and headache Chair: Karl Messlinger, Institute of Physiology & Exp. Pathophysiology, University of Erlangen-Nu¨rnberg, Germany Role of neuropeptides in primary headaches – current concepts K. Messlinger, Institute of Physiology & Exp. Pathophysiology, University of Erlangen-Nu¨rnberg, Germany TRPV1-mediated CGRP release from skin, nerve and dura mater, in vitro S.K. Sauer, M.J.M. Fischer, P.W. Reeh, Department of Physiology and Exp. Pathophysiology, FAU Erlangen, Germany On the mechanism of CGRP release from rat cranial dura R.W. Carr, K. Messlinger, Institute of Physiology & Exp. Pathophysiology, University of ErlangenNu¨rnberg, Germany Nociceptor sensitization by CGRP is a heritable trait in mice and occurs in cultured rat DRG neurons K. Zimmermann, M. Gautam, P.W. Reeh, Institute of Physiology & Exp. Pathophysiology, Erlangen, Germany

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The non-peptide CGRP receptor antagonist BIBN4096BS lowers the activity of neurons with meningeal input in the rat spinal trigeminal nucleus M.J.M. Fischer, S. Koulchitsky, K. Messlinger, Institute of Physiology & Exp. Pathophysiology, Erlangen, Germany

Preclinical and clinical profiling of the first non-peptide CGRP antagonist BIBN4096 K. Arndt, S. Just, K.A. Petersen, H. Doods, BoehringerIngelheim Pharma GmbH & Co. KG, Department of CNS Research, D-88397 Biberach, Germany

Role of neuropeptides in primary headaches – current concepts K. Messlinger, Institute of Physiology & Exp. Pathophysiology, University of Erlangen-Nu¨rnberg, Germany E-mail: [email protected] Sensory neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) have long been known as mediators of neurogenic inflammation that are involved in the pathophysiological processes of hyperalgesia and pain. Neurogenic inflammation has also been hypothesized to underlie spontaneous attacks of primary headaches such as migraine pain and cluster headache. This idea was based on animal experiments in which substance P caused plasma protein extravasation and CGRP caused arterial vasodilation in the cranial meninges. Whereas the occurrence of neurogenic plasma extravasation in primary headaches is now controversially discussed, the functions of CGRP attract more and more interest. CGRP is produced from a large proportion of nociceptive trigeminal afferent neurons. During attacks of migraine and other primary headaches, CGRP concentrations have been found to be increased in the plasma of the internal jugular vein, indicating that CGRP is released from intracranial afferents (Edvinsson and Goadsby, 1998). Infusion of CGRP has been shown to cause headaches in migraineurs suggesting that CGRP can induce or facilitate nociceptive processes in the trigeminal system (Lassen et al., 2002). In a clinical trial the first non-peptide CGRP receptor antagonist BIBN4096BS proved to be therapeutically effective in a group of migraineurs (Olesen et al., 2004). These clinical observations raise the question, by which mechanisms CGRP is released from afferent neurons and whether CGRP itself has a nociceptive function. In this symposium, the mechanisms and nociceptive functions of CGRP release in spinal and trigeminal tis-

sues will be reviewed based on some newly developed in vitro and in vivo preparations. The role of capsaicin (TRPV1) receptor channels in CGRP release from different tissues and the importance of afferent activation and calcium inflow in meningeal nociceptors will be discussed. Sensitizing effects of CGRP in heat-sensitive cutaneous nociceptors will be shown. Finally, a report will be given on pre-clinical pharmacological data and the clinical trial (Olesen et al., 2004) with the first effective non-peptide CGRP receptor antagonist, BIBN4096BS. References Edvinsson, L., Goadsby, P.J., 1998. Eur. J. Neurol. 5, 329–341. Lassen, L.J., et al., 2004. Cephalalgia 22, 54–61. Olesen, J., et al., 2004. New Engl. J. Med. 350, 1104–1110.

TRPV1-mediated CGRP release from skin, nerve, dura mater, in vitro S.K. Sauer, M.J.M. Fischer, P.W. Reeh, Department of Physiology and Exp. Pathophysiology, FAU Erlangen, Germany E-mail: [email protected] Aim of investigation. The TRPV1 receptor is the polymodal integrator for noxious chemical and heat stimuli in the sensory nociceptive system. Activation of the TRPV1 cause neuropeptide release from nociceptive afferents in various tissues, which can be used as a measure for nociceptive transduction. Here we present comparative data from different preparations on the TRPV1-mediated CGRP release and its sensitization, analogous to conditions that may underlie processes of neurogenic inflammation as discussed for migraine. Methods. CGRP release was measured, in vitro from excised skin flaps, desheathed sciatic nerves or hemisected skulls with intact dura mater in rat and mouse. CGRP release was stimulated by heat, acidic buffer and capsaicin. CGRP content of the eluates was determined using an enzyme immuno-assay kit (SPIbio, France). Results. In all three preparations protons and heat evoked Ca++-dependent CGRP release. In the sciatic nerve proton (pH 5.2)-induced release could partially be blocked by the TRPV1 receptor antagonists capsazepine and ruthenium red and in the TRPV1 )/) mice the release was completely lost. Same was observed in the skin and the dura. Heat (47 C) – induced CGRP release from the sciatic nerve could neither be blocked by the antagonists nor in TRPV1 )/) mice, whereas in the skin from TRPV1 )/) animals the heat-induced release was reduced by about 50%. Coadministration of PGE2 (10 lM) increased proton-evoked CGRP release in skin, nerve and dura, suggesting that CGRP release is facilitated by PKA activation. Activation of the PKC with the phorbol ester

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PMA also led to sensitization of heat-induced release in the skin and nerve preparation. Conclusion. Comparison of TRPV1-mediated CGRP release from different tissues suggests that besides TRPV1 other heat sensitive ion channels are involved in heat transduction, whereas proton responsiveness seems to be entirely dependent on TRPV1 activation. Under pathological conditions, in vivo when tissue acidosis or (elevated) body temperature may increase CGRP release and cause neurogenic inflammation, which in turn may induce a cascade of nociceptive processes that exaggerate pain states such as assumed for migraine attacks. Acknowledgement. Supported by the DFG-SFB 353.

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solution induced neither impulse activity nor CGRP release. Conclusion. Potassium (60 mM) induced CGRP release from rat dura is dependent on the presence of both extracellular calcium and sodium action potentials, but not sodium per se. Acknowledgement. R.C. was supported by the Alexander von Humboldt Foundation. References Spitzer, M.J., Sauer, S.K., Reeh, P.W., 2004. Eur. J. Physiol. 447, S87. Viana, F., de la Pen˜a, E., Pecson, B., Schmidt, R.F., Belmonte, C., 2001. Eur. J. Neurosci. 13, 722–734.

On the mechanism of CGRP release from rat cranial dura R.W. Carr, K. Messlinger, Institute of Physiology and Exp. Pathophysiology, University of ErlangenNu¨rnberg, Germany E-mail: [email protected]

Nociceptor sensitization by CGRP is a heritable trait in mice and occurs in cultured rat DRG neurons K. Zimmermann, M. Gautam, P.W. Reeh, Institute of Physiology & Exp. Pathophysiology, Erlangen, Germany E-mail: [email protected]

Aim of investigation. Intracellular calcium stores in small diameter sensory afferents are distributed scantily. Accordingly, the release of CGRP from sensory axons has been shown to depend on extracellular calcium influx through either calcium selective channels (Spitzer et al., 2004) or perhaps through calcium permeable transduction channels (e.g., TRPV1, ANKTM1 and mechanotransducers, Viana et al., 2001). For some axonal functions such as the axon reflex, neuropeptide release must also involve sodium dependent action potentials. Consequently, we investigated the role of sodium action potentials in CGRP release from rat dura. Methods. Rats were killed by CO2 inhalation and decapitated. The heads were bi-sected mid-sagitally and the cortex and brain stem removed. The remaining dura lined half-skull was warmed to 37 C. The CGRP content of 500 ll aliquots instilled into the half-skull for periods of 5 min was determined by ELISA. In some experiments nerve activity was recorded from a portion of the spinosus nerve. The standard physiological solution contained (in mM) 112 NaCl; 0.69 MgSO4; 1.67 KH2PO4; 1.85 C6H11O7.K; 5.05 D (+)-glucose; 7.6 D (+)-sucrose; 10 HEPES, adjusted to pH 7.3. For sodium replacement, an equimolar amount of either LiCl or choline–chloride was substituted for NaCl. Results. In standard physiological solution, a depolarizing potassium (60 mM) stimulus induced activity in sensory fibres innervating the dura and, in the presence of extracellular calcium, CGRP release. TTX (1 lM) reduced both impulse activity and the amount of CGRP released by 60 mM potassium. Impulse activity and CGRP release were also evoked by 60 mM potassium when sodium was replaced with lithium. However, when sodium was replaced with choline, 60 mM potassium

Aim of investigation. Neuropeptides released from primary afferents could act through autoreceptors to sensitize nociceptors and contribute to pain and hyperalgesia as has been previously proposed for migraine. Attempts to reveal such a vicious circle have failed, perhaps because an as yet unknown pathophysiological condition has been missing and/or the appropriate animal model has not been used. Methods. In comparison to C57BL/6, AKR albino mice are much less sensitive to noxious heat at both the behavioural and neuronal level as well as they express and release much less CGRP. Using single-fibre recordings and whole-cell patch-clamping of DRG neurons, we assessed the heat responsiveness of sensory neurons before and after CGRP administration [10-6M]. In Wistar rats the effects of a competitive CGRP receptor blocker, and a PKA inhibitor, H89 were investigated using DRG cells cultured in presence of NGF. Results. In both preparations AKR mice regularly exhibited poor heat responsiveness, with high thresholds and small heat-activated currents (Iheat) or numbers of action potentials, respectively. By CGRP almost all AKR neurons were significantly sensitized. In contrast, C57BL/6 mice showed pronounced heat responses in both preparations but no significant CGRP-induced sensitization to heat. In the rat single-fibre experiments CGRP caused dramatic sensitizations to heat, but only in 3 of 27 C-fibres. However, the majority of the rat DRG neurons (56) showed a significant, time and concentration dependent sensitization to heat after CGRP with a large increase in Iheat but only a small decrease in the heat thresholds, whereas the other neurons displayed the usual desensitization upon repeated heat stimulation. Both the CGRP antagonist and H89 abolished the

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CGRP effect, suggesting that a CGRP receptor-G protein mediated activation of PKA is involved. Conclusion. Noxious heat sensitivity and sensitizability by CGRP as well as expression of and sensitivity to CGRP are all congenic heritable traits that are linked to one another by the acute and sustained sensitizing effects of CGRP on nociceptive neurons that is also present under artificial culture conditions (in rat neurons). Thus, a vicious circle may evolve in peripheral nociception under certain pathophysiological conditions that appear worthwhile to be uncovered. Acknowledgement. Supported by DFG-SFB353/B12 and the German-Israeli-Foundation.

The non-peptide CGRP receptor antagonist BIBN4096BS lowers the activity of neurones with meningeal input in the rat spinal trigeminal nucleus M.J.M. Fischer, S. Koulchitsky, K. Messlinger, Institute of Physiology & Exp. Pathophysiology, University of Erlangen-Nu¨rnberg, Germany E-mail: [email protected] Aim of investigation. Calcitonin gene-related peptide (CGRP) is suggested to play a major role in the pathogenesis of migraine and other primary headaches. CGRP is increased in the cranial circulation during migraine attacks and released from trigeminal afferents under experimental conditions. In a rat model of meningeal nociception we examined the effect of the new non-peptide CGRP receptor antagonist BIBN4096BS on the discharge activity in neurones of the spinal trigeminal nucleus (STN) with meningeal input. STN neurones integrate nociceptive afferent inputs from trigeminal tissues including intracranial afferents. The activity of STN neurons is thought to reflect the trigeminal nociceptive activity that appears as facial pain and headache in man. Methods. Single cell activity in the STN was recorded. All units had receptive fields located in the exposed parietal dura mater. Heat and cold stimuli were repetitively applied in a fixed pattern of ramp and step modes to the dura to evoke neuronal activity. BIBN4096BS dissolved in physiological saline was i.v. infused at cumulative doses of 300 and 900 lg/kg. In other experiments BIBN4096BS was topically applied onto the exposed dura at a concentration of 10–3 M. Results. The lower dose of i.v. BIBN4096BS reduced spontaneous activity by more than 30%, the higher dose to 50% of the control level, while saline had no effect. The activity evoked by heat ramps was reduced after i.v. BIBN4096BS by about 50%. Topical administration of BIBN4096BS did not significantly change the neuronal activity within 15 min. Conclusion. We conclude that CGRP significantly contributes to the maintenance of spontaneous impulse

activity in neurons of the STN. Blockade of CGRP receptors, possibly at central and peripheral sites, may therefore be an effective way to decrease nociceptive transmission, thereby offering new therapeutic strategy for the treatment of facial pain and primary headaches.

Preclinical and clinical profiling of the first non-peptide CGRP antagonist BIBN4096 K. Arndt, S. Just, K.A. Petersen, H. Doods, Department of CNS Research, Boehringer-Ingelheim Pharma GmbH & Co. KG, D-88397 Biberach, Germany E-mail: [email protected] During migraine attacks, plasma levels of the potent vasodilator calcitonin gene-related peptide (CGRP) are elevated in migraine patients. Would a drug that counteracts CGRP function during an acute attack relief the pain? Here, we present preclinical and clinical evidence that the first non-peptide high-affinity antagonist BIBN4096 potently reverses CGRP-induced changes in peripheral hemodynamics in animal models and human studies, and that BIBN4096 alleviates acute migraine pain in migraineurs (Olesen et al., 2004). In the animal models, hemodynamic changes were induced by either CGRP injection or electrical stimulation of the trigeminal ganglion and facial blood flow changes (FBF) were monitored (Doods et al., 2000). Here, BIBN4096 fully reversed the evoked vasodilation with ED50s of 0.003 and 0.05 mg/kg in marmoset monkeys and rats, respectively. In healthy volunteers, a 2.5 mg dose of BIBN4096 significantly attenuated human?-CGRP-induced increase in cerebral blood flow. In a multicenter, double blind, placebo-controlled, randomized clinical trial the same dose relieved headache pain 2 h after start of treatment in 65.6% of migraine patients (versus 26.8% placebo), and 43.8% of the patients (versus 2.4% placebo) reported to be pain free. Together, our data demonstrate that counteracting CGRP function during a migraine attack successfully alleviates migraine headache.

References Olesen, J., et al., 2004. NEJM 350(11), 1104ff. Doods, H., et al., 2000. Br. J. Pharmacol. 129(3), 420ff.

Nociceptin/Orphanin FQ and its receptors-novel ligands and therapeutic opportunities Chair: Girolamo Calo` Pharmacology, University of Ferrara, Italy How does the nociceptin/orphanin FQ receptor bind peptides?

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Jean-Claude Meunier, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, France Novel potent peptide ligands for the nociceptin/orphanin FQ receptor John McDonald, University Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group) Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, UK Blocking Nociceptin/orphanin FQ signalling produces antidepressant like effects in rodents Girolamo Calo´, Pharmacology, University of Ferrara, Italy Nociceptin/orphanin FQ control of reward: pharmacological and genetic studies Niall P. Murphy, RIKEN Brain Science Institute, Japan Nociceptin/orphanin FQ receptor antagonists reverse akinesia in parkinsonism models Michele Morari, Department of Experimental and Clinical Medicine, Section of Pharmacology, and Neuroscience Center, University of Ferrara, Italy How does the nociceptin/orphanin FQ (NOP) receptor bind peptides J.-C. Meunier, B. Bes, C.M. Topham, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France E-mail: [email protected] Aim of investigation. Provide a stuctural framework for the understanding of the differential binding modes of the heptadecapeptide nociceptin and of hexapeptides to the NOP receptor. Methods. Photo-affinity labeling of the receptor using photolabile derivatives of nociceptin and hexapeptides, and molecular modeling of the receptor in complex with nociceptin and hexapeptides. Results. (1) The UV-activated nociceptin derivative [Bpa10, 125I-Tyr14]-noc/oFQ labels the NOP receptor sequence [296-302], comprising the C-terminus of extracellular loop 3 and the N-terminus of transmembrane helix VII. (2) the UV-activated hexapeptide derivative Ac-Arg-Bpa-(125I-Tyr)-Arg-Trp-Arg-NH2 ([Bpa2, 125I-Tyr3]-HP1), labels the NOP receptor sequence [107-113] region of the ORL1 receptor at the C-terminus of transmembrane helix II. This region is not only widely separated in one-dimensional amino acid sequence space from the [Bpa10, 125I-Tyr14]-noc/

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oFQ photo-reactive [296–302] residue fragment, but the two regions are also distant in Euclidean space in the modelled receptor. The results suggest distinct modes of interaction of the hexapeptide and nociceptin with the NOP receptor, consistent with their distinct pharmacological activities. Using the recently published 2.8 resolution crystallographic structure of bovine rhodopsin as a more accurate template for receptor modelling, we compare possible binding modes of the hexapeptide and nociceptin to the NOP receptor. Binding of [Bpa2, 125I-Tyr3]-HP1 at the extracellular surface of the receptor parallel to the membrane plane with the reactive Bpa2 ketone orientated towards atoms of the Leu112 side-chain in the photo-reactive region permits extensive overlap with the proposed nociceptin binding site, notably around a turn in the second extracellular loop carrying five negatively charged amino acid residue sidechains. Conclusions. Collectively, the present results provide a stuctural framework for the understanding of the differential binding modes of the hexapeptides and nociceptin to the NOP receptor, and suggests novel strategies for designing novel NOP receptor ligands, especially ligands that will not interact with the receptor’s transmembrane binding pocket.

Novel potent peptide ligands for the nociceptin/orphanin FQ receptor John McDonald a, Tim A. Barnes a, Remo Guerrini b, Severo Salvadori b, David G. Lambert a, a University Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE1 5WW, UK, b Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy E-mail:[email protected] Aim. Previous structure activity relationship (SAR) studies have showed that the potency of nociceptin/orphanin FQ (N/OFQ), at the N/OFQ receptor (NOP) is increased through addition of fluorine to the Phe4 residue, or by the incorporation of two basic residues, Arg14-Lys15 to the C-terminal portion of the peptide [Arg14,Lys15]N/OFQ. Further SAR studies show that N/OFQ efficacy is altered through reduction of the peptide bond between Phe1 and Gly2 [Phe1?(CH2NH)Gly2]N/OFQ(1-13)-NH2, or shifting the Phe1 side chain on the N-terminal nitrogen, [Nphe1]N/OFQ(1-13)-NH2. The aim of the study was to look at the effects of combinations of these modifications (12 peptides) on the affinity, functional potency and efficacy at NOP receptors.

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Methods. CHO cells expressing the human NOP receptor (CHOhNOP) were used to study ligand affinity ([leucyl-3H]N/OFQ binding studies), and functional potency/efficacy (GTPg[35S] and cAMP assays). Results. All peptides behaved as NOP ligands; the chemical modifications Arg14-Lys15 and (pF)Phe4 and especially their combination produced a clear increase of ligand affinity/potency. Those peptides with normal Phe1-Gly2 peptide bonds displayed full agonist activity, those with the Phe1?(CH2NH)Gly2 modification behaved as partial agonists of varying efficacy, while those with the Nphe1 modification as either partial agonists or pure antagonists. Conclusions. Of the 12 N/OFQ-NH2 analogs created three molecules show particularly interesting profiles; i.e., the agonist [(pF)Phe4Arg14Lys15]N/OFQ-NH2, the partial agonist [Phe1?(CH2NH)Gly2 (pF)Phe4Arg14Lys15]N/OFQ-NH2 and the pure antagonist [Nphe1Arg14Lys15]N/OFQ-NH2.

Blocking N/OFQ signalling produces antidepressant like effects in rodents E.C. Gavioli a,b, C.W. Vaughan c, G. Marzola a, R. Guerrini d, V.A. Mitchell c, S. Zucchini a, T.C.M. De Lima b, G.A. Rae b, S. Salvadori d, D. Regoli a, G. Calo’ a, a Pharmacology, University of Ferrara, Italy, b Pharmacology, University of Santa Catarina, Brazil, c Pain Institute, University of Sydney, Australia, d Pharmaceutical Sciences, University of Ferrara, Italy E-mail: [email protected] Aim of investigation. Studies using receptor antagonists and knockout animals show that blockade of nociceptin/orphanin FQ (N/OFQ)-NOP receptor signalling causes antidepressant-like effects in mice submitted to the forced swimming test (FST). The present study aimed to further explore the antidepressantlike properties of the NOP antagonist UFP-101 across species (mouse and rat) and assays (FST and tail suspension test (TST)), and to identify some of the mechanisms involved in its actions. Results. UFP-101 (10 nmol, i.c.v.) reduced immobility time of Swiss mice in the TST (from 179 ± 11 s to 111 ± 10 s). N/OFQ (1 nmol, i.c.v.) was without effect per se, but fully prevented the effect of UFP-101. Spontaneous immobility time of NOP–/– CD1-C57BL/ 6J-129 mice in the TST was much lower than that displayed by their wild-type NOP+/+ littermates (75 ± 11 s vs 144 ± 17 s) or by Swiss mice. UFP-101 (10 nmol, i.c.v.) decreased immobility time (–65%) and increased climbing time (71%) in rats submitted to the FST. In rat brain slices, N/OFQ (100 nM) triggered robust K+-dependent hyperpolarizing currents in locus coeruleus and dorsal raphe neurones. UFP-101 (3 lM) fully prevented N/OFQ-induced currents, being in-

active per se. Fluoxetine, desipramine (both 30 mg/kg, i.p.) and UFP-101 (10 nmol, i.c.v.) reduced mouse immobility time in the mouse FST. The serotonin synthesis inhibitor PCPA (4 · 100 mg/kg/day i.p.) prevented the antidepressant-like effects of fluoxetine and UFP-101 (but not desipramine), whereas DSP-4 (neurotoxic for noradrenergic neurones; 50 mg/kg, i.p., 7 days beforehand), suppressed only the effect of desipramine. Neither pretreatment affected spontaneous immobility time per se. Conclusion. UFP-101 exhibits pronounced antidepressant-like effects in distinct species and animal models, possibly by preventing the inhibitory effects of endogenous N/OFQ on brain monoaminergic (in particular serotonergic) neurotransmission. Participation of the N/OFQ-NOP receptor system in mood modulation sets new potential targets for antidepressant drug development.

Nociceptin/orphanin FQ control of reward: pharmacological and genetic studies N.P. Murphy a, C. Okabe a, M. Koizumi a, N. Midorikawa a, H. Takeshima b, K. Sakoori a, a RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, b Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan E-mail: [email protected] Aim of investigation. Several lines of evidence indicate that administration of nociceptin/orphanin FQ (N/ OFQ) is able to reverse the rewarding and incentive properties of several commonly abused drugs including opiates, psychostimulants and alcohol. However, a role, if any, for endogenous N/OFQ in the control of these behaviors and their underlying neurobiology is yet to be determined. Methods. A combination of molecular (mapping of c-fos expression as an index of neural activity) and behavioral (place conditioning, behavioral sensitization, bottle choice) techniques was used. The effects of administration of the N/OFQ antagonist UFP-101 and null mutation of the N/OFQ receptor on basal and stimulated changes in hedonic state, and the response to chronic drug treatment were determined. Results. Central administration of UFP-101 induced a mild conditioned place preference, and enhanced the conditioned place preference for methamphetamine. Mildly enhanced place preferences for methamphetamine were also found in N/OFQ receptor knockout mice, whereas the aversive effects of lithium chloride, naloxone and the kappa opioid receptor antagonist U50488H were little changed. Studies using a bottle choice paradigm showed N/OFQ receptor knockout mice exhibited a decreased preference for ethanol, particularly when ethanol was presented at higher

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concentrations. Behavioral sensitization to methamphetamine in a novel environment was little affected by null mutation of the N/OFQ receptor, though N/OFQ knockout mice generally showed diminished acute responses to methamphetamine but enhanced sensitized responses in terms of c-fos expression in several elements of the extended amygdala. Conclusion. Endogenous N/OFQ contributes to the regulation of basal and drug stimulated changes in hedonic state, and some of the long-term conditioned effects of abused drugs. Acknowledgment. This work was funded by the RIKEN Brain Science Institute.

Nociceptin/orphanin FQ receptor antagonists reverse akinesia in rat parkinsonism models M. Marti a, F. Mela a, M. Fantin a, R. Guerrini b, C. Bianchi a, M. Morari a, a Department of Experimental and Clinical Medicine, Section of Pharmacology, and Neuroscience Center, University of Ferrara, Ferrara, Italy, b Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy E-mail: [email protected] Aims of investigation. Endogenous nociceptin/orphanin FQ (N/OFQ) inhibits the nigrostriatal dopaminergic (DAergic) transmission and motor behaviour via activation of (NOP) receptors located in the substantia nigra reticulata (SNr) (Marti et al., 2003). Indeed, SNr injection of NOP receptor antagonists elevated striatal DA release and rotarod performance in rats. The present study was undertaken to investigate whether NOP receptor antagonists reverse akinesia in parkinsonism models and to study the mechanisms involved. The role of endogenous N/OFQ in modulating parkinsonism was further studied in NOP receptor knockout (NOP–/–) mice. Methods. Antiakinetic effects of UFP-101 and J-113397 were measured in rats made cataleptic with haloperidol or hemiparkinsonian with 6-hydroxydopamine. Antiakinetic effects were assessed by behavioural tests (the bar and the rotarod tests) while mechanisms involved were investigated by monitoring SNr GLU release in microdialysis experiments. Results. The bar test showed that SNr UFP-101 injections (30 nmol) reversed haloperidol-induced catalepsy. The rotarod test showed that SNr injections of UFP-101 (0.1–10 nmol) or J-113397 (0.1–1 nmol) or i.p. administration of J-113397 (0.1 and 1 mg/kg) relieved akinesia in hemiparkinsonian rats. Motor stimulating effects of both antagonists were greater and induced at lower doses in hemiparkinsonian than nave rats. Antiparkinsonian actions of both antagonists were associated with inhibition of nigral GLU release. A role for

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N/OFQ in sustaining parkinsonism was further supported by the findings that NOP–/– were partially resistant to haloperidol-induced catalepsy. Conclusion. Endogenous N/OFQ in the SNr contributes to sustain parkinsonian symptoms. NOP receptor antagonists may represent a new approach for management of parkinsonism. Acknowledgements. Cofin 2002 and FIRB Grants to C.B. Reference Marti M, Mela F. Ulazzi L. Vaccari E. Guerrini R, Trapella C. Beani L, Bianchi C, Morari M. VIII Monothematic Meeting: ‘‘Nociceptin/Orphanin FQ and its receptor’’. September 15th 2003, Camerino, Italy.

Opioid receptor-like 1 stimulation in the collecting duct induces aquaresis through vasopressin-independent aquaporin-2 down-regulation N. Hadrup a, J.S. Petersen b, T.E.N. Jonassen a, a Department of Pharmacology, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, b Zealand Pharma A/S, Smedeland 26 B, DK2600 Glostrup, Denmark E-mail: fi[email protected] Nociceptin, the endogenous ligand of the inhibitory G-protein coupled opioid receptor-like 1 receptor produces aquaresis (i.e., increases the excretion of solute free urine) in rats. However, the mechanism underlying this effect has not yet been explained. Using immunohistochemistry we have found that the opioid receptor-like 1 receptor in the rat kidney, co-localized with the vasopressin regulated water channel aquaporin-2 in inner medullary collecting ducts. We investigated the aquaretic effect of opioid receptor-like 1 receptor stimulation by infusing the selective nociceptin analogue ZP120C; volume depletion was prevented by computerdriven, servo-controlled i.v. volume replacement with 50 mM glucose. ZP120C induced a marked and sustained aquaresis, in normal and congestive heart failure rats, in absence of changes in vasopressin plasma concentrations. The ZP120C induced aquaresis was associated with down-regulation of the aquaporin-2 protein level in both rat groups suggesting that opioid receptor-like 1 receptor stimulation produces aquaresis by inhibiting the vasopressin type-2 receptor mediated stimulation on collecting duct water reabsorption. However, substantial amounts of PKA-mediated serine-256 phosphorylated aquaporin-2 were still present after 4 h of ZP120C treatment. Furthermore, neither preincubation with nociceptin nor ZP120C inhibited vasopressinmediated cAMP accumulation in isolated collecting ducts. We conclude that renal opioid receptor-like 1

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receptor stimulation, in normal and congestive heart failure rats, produces aquaresis by a direct renal effect, via aquaporin-2 down regulation, through a mechanism not involving inhibition of vasopressin type-2 receptor mediated cAMP production.

Somatostatin and cortistatin in the human immune system Leo J. Hofland, Departments of Internal Medicine, and Immunology, Erasmus MC, Rotterdam, The Netherlands

Neuropeptides in immunity Co-chairs: Illana Gozes a, Mario Delgado b, a Sackler Med. School, Tel Aviv University, Israel, b Institute of Parasitology and Biomedicine-CSIC, Granada, Spain

VIP: a ‘‘very important peptide’’ in immunomodulation. Potential therapeutic applications Mario Delgado, Institute of Parasitology and Biomedicine, CSIC, Granada, Spain E-mail: [email protected]

Neuropeptide signatures in immune functions and disfunctions Eberhard Weihe, Department of Molecular Neuroscience, Philipps-University, Marburg, Germany VIP: a ‘‘very important peptide’’ in immunomodulation. Potential therapeutic applications Mario Delgado, Institute of Parasitology and Biomedicine, CSIC, Granada, Spain VIP decreases the expression of Toll-like receptors 2 and 4 in a mice model of Crohn’s disease R.P. Gomariz, A. Arranz, C. Abad, M. Garcia, C. Martinez, F. Rosignoli, M. Torroba, J. Leceta, M.G. Juarranz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain Expression and function of substance P in non-neuronal cells correlated with infection and immunity J.P.Stewart c, A. Kipar d, J.P. Quinn a,b, a Department of Physiology, The University of Liverpool, UK, b Department of Human Anatomy and Cell Biology, The University of Liverpool, UK, c Departments of Medical Microbiology, The University of Liverpool, UK, d Faculty of Veterinary Sciences, The University of Liverpool, UK PACAP, VIP and cytokines: reciprocal immune/neural interactions after nerve injury James A. Waschek a, Brian Armstrong a, Catalina Abad b, Rosa P. Gomariz b, Seririthanar Chhith a, Gardenia Cheung-Lau a, a University of California at Los Angeles, Los Angeles, CA, USA, bUniversidad Complutense de Madrid, Madrid, Spain VIP induces tolerogenic differentiation of immune cells J. Leceta, C. Martinez, M.G. Juarranz, C. Abad, M. Delgado, M. Garcia, R.P. Gomariz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain

Recent reports identified neural pathways, both hard-wiring and soluble mediators, that control and adjust the peripheral immune response. Immune organs are innervated by fibers rich in neurotransmitters and neuropeptides, that are released in inflammatory conditions. Here I focus on the immunomodulatory role of the vasoactive intestinal peptide (VIP). VIP is a neuropeptide present and released from both innervation and immune cells, particularly Th2 cells, and immune cells express receptors for VIP. VIP has a general anti-inflammatory effect, both in innate and adaptive immunity. In innate immunity, VIP inhibits the production of pro-inflammatory cytokines and chemokines from macrophages, microglia and dendritic cells. In addition, VIP reduces the expression of costimulatory molecules on the antigen-presenting cells, and therefore reduces stimulation of antigenspecific CD4+ T cells. In terms of adaptive immunity, VIP promotes Th2-type responses, and reduces the pro-inflammatory Th1-type responses. VIP is rapidly transforming into something more than a mere neuropeptide. In evolving scientifically from a neuropeptide to a novel agent for modifying immune function and, possible a cytokine-like molecule, VIP research has engaged many physiologists, molecular biologists, biochemists, endocrinologists and pharmacologist and it is a paradigm to explore mutual interactions between neural and neuroendocrine links in health and disease. Recognition of the central functions VIP plays in cellular processes is focusing our attention on this ‘‘very important peptide’’ as exciting new candidates for therapeutic intervention and drug development.

VIP decreases the expression of Toll-like receptors 2 and 4 in a mice model of Crohn’s disease R.P. Gomariz, A. Arranz, C. Abad, M. Garcia, C. Martinez, F. Rosignoli, M. Torroba, J. Leceta, M.G. Juarranz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain E-mail: [email protected]

Abstracts / Neuropeptides 38 (2004) 385–424

Aim of investigations. Toll-like receptors (TLRs) belong to pattern recognition receptor family (PRRs) and play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components, being important connectors between the innate and adaptative immunity. The activation of TLRs, induce the production of proinflammatory and Th1 inflammatory mediators in immune cells. Vasoactive intestinal peptide (VIP) is a neuropeptide present in nerve fibers and lymphocytes that modulates innate and adaptative immunity (Gomariz et al., 2001). In inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) an excessive immune response leads to the production of proinflammatory factors and recent data suggest that CD reflect an excessive Th1 response. We have demonstrated that treatment with VIP reduced the clinical and histopathological severity of TNBS-induced colitis by decreasing both inflammatory and Th1-driven autoimmune components of the disease (Abad et al., 2003), but nothing is known about the putative involvement of TLRs both in the disease and healing. The aim of the present work is to study both the expression of TLR2 and TLR4 receptors along the TNBS-induced colitis (an animal model of Crohn’s disease) and the possible involvement of them in the therapeutic effect of VIP. Methods. A 3-mg enema of TNBS was given to BALB/c mice, and VIP (1 nmol) was given every other day. RNA and membrans from colon were obtained at different days after TNBS induction (1, 3, 5 and 7 days). mRNA expression for TLR2 and TLR4 was quantified by real time PCR. Protein expression was quantified by immunoblotting using specific antibodies against mouse TLR2 and TLR4. Results. Our results showed the constitutive expression of TLR2 and TLR4, as well as the up-regulation of TLR2 and TLR4 in the colon after TNBS administration. VIP treatment significantly decreased TNBS-induced TLR2 and TLR4 mRNA expression and thus, their protein levels in this tissue. Conclusion. The present study demonstrates that the modulation of TLRs receptors is involved in the beneficial effects of VIP in an animal model of Crohn’s disease. Acknowledgement. This work was supported by the Spanish Department of Science and Technology Grant BFI 2002-03489.

References Gomariz, R.P., Martinez, C., Abad, C., Leceta, J., Delgado, M:, 2001. Immunology of VIP: a review and therapeutical perspectives. Current Pharmaceutical Design 7, 89–111. Abad, C., Martinez, C., Juarranz, M.G., Arranz, A., Leceta, J., Delgado, M., Gomariz, R.P., 2003. Therapeutic effects of

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vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn’s disease. Gastroenterology 124, 961–71.

Expression and function of substance P in non-neuronal cells correlated with infection and immunity J.P.Stewart c, A. Kipar d, J.P. Quinn a,b, a Department of Physiology, The University of Liverpool, UK, b Department of Human Anatomy and Cell Biology, The University of Liverpool, UK, cDepartments of Medical Microbiology, The University of Liverpool, UK, d Faculty of Veterinary Sciences, The University of Liverpool, UK E-mail: [email protected] Aim. We have demonstrated that the tachykinins control previously considered cytokine regulated responses during both osteoarthritis and host response to virus infection in the lung (Millward-Sadler et al., 2003; Payne et al., 2001). We now determine the cell type expressing the tachykinins and the time course over which this expression is induced and relate this to function. In particular we concentrated on tachykinin expression in airway surfaces in response to the herpesvirus MHV-68 which may determine whether the animals exhibits alveolar fibrosis to infection rather than a normal immune response to clear the virus. Methods. We used analysis of marker gene expression supported by a transgenic animal expressing a yeast artificial chromosome for the human Preprotachykinin A in which was embedded LacZ (MacKenzie et al., 2000) complemented by immunohistochemistry. These animals were infected with MHV-68 and substance P expression and lung pathology addressed over the time course of infection. Results and Conclusion. Tachykinin expression and peptide was found to be constitutively expressed in a variety of cells in both the lung and spleen and that this can be further induced by exposure to the virus very early in infection. The genetic background of the animal may determine in part this expression pattern. The induced expression of tachykinins in the lung is early response to virus infection and perhaps one of the initial mediators of the immune cascade which follows. References Millward-Sadler, S.J., Mackenzie, A., Wright, M.O., et al., 2003. Tachykinin expression in cartilage and function in human articular chondrocyte mechanotransduction. Arthritis Rheum 48, 146–156. Payne, C.M., Heggie, C.J., Brownstein, D.G., Stewart, J.P., Quinn, J.P., 2001. Role of tachykinins in the host response to murine gammaherpesvirus infection. J Virol 75, 10467–10471. MacKenzie, A., Payne, C., Boyle, S., Clarke, A.R., Quinn, J.P., 2000. The human preprotachykinin-a gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS. Mol Cell Neurosci 16, 620–630.

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VIP induces tolerogenic differentiation of immune cells J. Leceta, C. Martinez, M.G. Juarranz, C. Abad, M. Delgado, M. Garcia, R.P. Gomariz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain E-mail: [email protected]

genic challenge. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by the deregulated expression of many proinflammatory cytokines including tumour necrosis factor a´? (TNF-a´), with increased yet insufficient production of anti-inflammatory cytokines including IL-10. Several studies in animal models revealed that the T helper cell type 1 (Th1) derived cytokines profile predominates at the induction and acute phases of the disease, whereas T helper cell type 2 (Th2) mediated responses are associated with the remission phase of the disease suggesting a pathogenic role of Th1derived cytokines. Vasoactive intestinal peptide (VIP) is a neuropeptide that is released by nerve fibres and lymphocytes in the lymphoid microenvironment and modulates innate and adaptive immunity through cellular signalling mediated by G protein-coupled receptors. VIP has been clearly identified as a potent anti-inflammatory factor, and preferentially induces differentiation toward a Th2 response following antigen stimulation. In a recent report, by using collageninduced arthritis (CIA), a murine experimental model induced by immunization with type II collagen (CII), we have demonstrated that treatment of arthritic mice with VIP decreases the frequency of arthritis, delaying the onset, reducing the severity of arthritic symptoms, and preventing joint damage. The aim of this study was to determine the effect of VIP on the expression of cytokines, chemokines, and their receptors in joints of arthritic mice, as well as to check the immune response against CII of cells from treated mice. Methods. Detection of gene expression for cytokines, chemokines and their receptors were performed with microarrays using a mouse inflammatory cytokine/receptor GEArraysTM Q series Kit (Cat. No. mGEA1013030, Superarray, Bethesda, MD). To study the immune response of mice against CII we have detected the proliferative response and secretion of cytokines in cultures of synovial and spleen cells stimulated with CII. Results. Although the expression of cytokines and chemokines are greatly inhibited in the joints of arthritic mice treated with VIP, the high affinity receptor for IL-2 (IL-2Ra) is little affected, and the chemokine receptor CCR3 is upregulated. After VIP treatment immune cells show antigen-specific inhibition of proliferation, increased production of IL-4 and IL-10, and decreased production of IFN. Conclusion. VIP biases immune response toward a Th2/T regulatory reactivity.

Aim of investigation. There is an increasing body of evidence that the nervous system is capable of modulating the immune response. Neuropeptides have been shown to play a role in modulation of immune and inflammatory reactions being crucially involved in both conditioning and fine-tuning the host response to anti-

Hot topics and last advances Co-chairs: Daniel Hoyer a, John Taylor b, aNovartis Institutes for Biomedical Research, Basel, Switzerland, b IPSEN Group, Milford, MA, USA

PACAP, VIP and cytokines: reciprocal immune/neural interactions after nerve injury James A. Waschek a, Brian Armstrong a, Catalina Abad b, Rosa P. Gomariz b, Seririthanar Chhith a, Gardenia Cheung-Lau a, a University of California at Los Angeles, Los Angeles, CA, USA, b Universidad Complutense de Madrid, Madrid, Spain E-mail: [email protected] Aim of investigation. VIP and PACAP are well known immunomodulators, as shown by numerous in vitro and in vitro studies, including experiments using mice which overexpress or lack receptors for these peptides. VIP and PACAP are both strongly upregulated in neurons in several nerve injury models, yet the mechanisms for their upregulation, and the functions of the peptides in nerve injury have not been determined. We hypothesized that an immune response is required for the upregulation of these peptides in injured neurons, and that the upregulation of these peptides, in turn, regulates the immune response at the site of injury. Methods. These questions were addressed using the facial nerve injury model in wild type and immunodeficient SCID mice and in mice with targeted mutations in the genes encoding VIP and PACAP. Results. VIP and PACAP gene expression was strongly induced in wild type mice by either facial nerve axotomy or by application of an inflammatory stimulus to the facial nerve. The axotomy-induced upregulation of PACAP, but not VIP, was blocked in SCID mice, but was restored in SCID mice previously given an infusion of mixed and CD4-elected splenocytes from wild type mice. These studies imply a critical role for lymphocytes in the upregulation of PACAP in injured neurons. Moreover, the response profile for several cytokines in injured nerves was altered in PACAP knockout mice compared to control mice. Conclusion. These studies demonstrate bidirectional signaling between neurons and immune cells involving cytokines and PACAP in mice in the context of nerve injury.

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Supported by IPSEN and Novartis Evidence that the Actions of Ghrelin on Feeding and Weight Gain are Mediated by a Novel Ghrelin Receptor H.A. Halem a, J.E. Taylor a, J.Z. Dong a, R. Datta a, T.L. Horvath b, M.D. Culler a, a IPSEN Group, Milford, MA, USA, b Yale Medical School, New Haven, CT, USA In vivo characterization of SRA880, a somatostatin sst1 receptor antagonist D. Hoyer, K. Dixon, C. Gentsch, A. Vassout, A. Enz, A. Jaton, C. Nunn, P. Schoeffter, P. Neumann, T. Troxler, P. Pfaeffli, Neuroscience Research, Novartis Institutes for BioMedical Research, WSJ 386/745, CH4002 Basel, Switzerland In vivo characterization of ACQ090, a somatostatin sst3 receptor antagonist D. Hoyer, A. Vassout, C. Gentsch, A. Enz, J.M. Vigouret, A. Jaton, P. Schoeffter, P. Kelly, A. Wrynn, K. Hurth, T. Troxler, Neuroscience Research, Novartis Institutes for BioMedical Research, WSJ 386/745, CH4002 Basel, Switzerland From VIP and PACAP to ADNP and NAP: New horizons in brain development and neuroprotection I. Gozes a, I. Pilzer a, R.A. Steingart a, D. Dangoor a, S. Rubinraut b, M. Fridkin b, DE Brenneman c, I. Divinski a, S. Furman a, a Clinical Biochemistry, Sackler Med. School, Tel Aviv Univ., Israel, b Organic Chemistry, Weizmann Inst., Rehovot, Israel; 3NICHD, Bethesda, MD, USA

Regulation and Function of the Tachykinins K. Haddley, S. Vasiliou, E. Spencer, M. Howard, J.P. Quinn, Department of Physiology and Human Anatomy and Cell Biology, The University of Liverpool, UK

Functional characteristics of peptide histidine-isoleucine (PHI) and peptide histidine-methionine (PHM) versus VIP and PACAP in vertebrate brain J.Z. Nowak a,b, A. Dejda a, J.B. Zawilska a,c, a Centre of Medical Biology and Microbiology, Polish Academy of Sciences, Lodz, Poland, b Department of Pharmacology, Medical University, Lodz, Poland, c Department of Pharmacodynamics, Medical University, Lodz, Poland

Evidence that the Actions of Ghrelin on Feeding and Weight Gain are Mediated by a Novel Ghrelin Receptor H.A. Halem, a J.E. Taylor, a J.Z. Dong a , R. Datta a, T.L. Horvath b M.D. Culler a, a IPSEN Group,

Milford, MA, USA, Haven, CT, USA

399 b

Yale Medical School, New

Background. Growth hormone (GH) secretagogues (GHS) are synthetic, unnatural peptides and nonpeptide molecules that stimulate GH secretion by activating the growth hormone secretagogue-1a (GHS-1a) receptor. Recently, a 28 amino acid, acylated peptide, predominately produced by the gastrointestinal tract, was isolated as the endogenous ligand for the GHS-1a receptor and was termed ‘‘ghrelin’’. The GHS-1a receptor is expressed at low levels in many tissues, but it is most strongly expressed hypothalamus. As expected, ghrelin has potent GH-releasing action mediated predominately via the hypothalamus, but it is also the first circulating hormone demonstrated to promote feeding and adiposity and to regulate energy homeostasis following either central or peripheral administration.. These findings have fostered the idea that analogs of ghrelin may be useful therapeutically for treating cachexic states. Conversely, compounds that inhibit ghrelin action may be useful for treating obesity, and, consequently, a great deal of effort is currently focused on finding antagonists of the GHS-1a receptor. Experimental. We have discovered an analog of full-length human ghrelin, BIM-28163, which fully antagonizes the GHS-1a receptor by binding to, but not activating the receptor. We further demonstrate that BIM-28163 blocks the activation of the GHS-1a receptor by ghrelin and inhibits ghrelin-induced GH secretion in vivo. Unexpectedly, however, BIM-28163 increased feeding and weight gain. These results strongly suggest the presence of an unknown ghrelin receptor that modulates ghrelin actions on feeding and weight control. Furthermore, BIM-28163 acts as an antagonist of ghrelin-induced Fos protein immunoreactivity (Fos-IR) in the medial arcuate nucleus (mARC), an area involved in the ghrelin modulation of GH secretion. However, in the dorsal medial hypothalamus (DMH), a region associated with regulation of food intake, both ghrelin and BIM-28163 act as agonists to upregulate Fos-IR. The observation that ghrelin and BIM-28163 have different efficacies in inducing Fos-IR in the DMH, and that concomitant administration of ghrelin and an excess of BIM-28163 results in the same level of Fos IR as BIM-28163 administered alone. Conclusion. These findings indicate that the actions of ghrelin to stimulate feeding/weight are mediated by a novel, non-GHS-1a receptor, and further imply that the GHS-1a may not be the appropriate target for antiobesity strategies. Acknowledgement. These studies were supported by IPSEN.

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In vivo characterization of SRA880, a somatostatin sst1 receptor antagonist D. Hoyer, K. Dixon, C. Gentsch, A. Vassout, A. Enz, A. Jaton, C. Nunn, P. Schoeffter, P. Neumann, T. Troxler, P. Pfaeffli, Neuroscience Research, Novartis Institutes for BioMedical Research, WSJ 386/745. CH4002 Basel, Switzerland Somatostatin (SRIF, somatotropin release inhibiting factor) acts via two families of G-protein coupled receptors: SRIF-1 (sst2, sst3, sst5) with high affinity for octreotide (SMS 201-995, Sandostatin), and SRIF-2 (sst1, sst4) with low affinity for Sandostatin (Hoyer et al, TiPS, 1995, 16: 86–88). Here we describe in vivo features of a selective sst1 receptor antagonist: SRA880 ([3R,4aR,10aR]-1,2,3,4,4a,5,10,10a-octahydro-6-methoxy-1-methyl-benz[g] quinoline-3-carboxylic-acid-4-(4nitro-phenyl)-piperazine-amide, hydrogen malonate) has high affinity (pKd’s = 8.0–8.2) and acts as an antagonist at native or recombinant sst1 receptors (SRIFinduced inhibition of forskolin-stimulated cAMP production, SRIF-stimulated GTPgS binding). SRA880 is orally bioavailable and brain penetrant. SRA880 was inactive in classical tests for anxiolytics and antidepressants. However, in aggressive mice model, the matched pairs and aggressive-resident encounters, SRA880 lowered high intensity elements of attacks, chases and bites in ‘‘residents’’. SRA880 also reversed the social withdrawal characteristic of ‘‘intruder’’ mice exposed to aggressive ‘‘residents’’. Thus, intruder mice regained their propensity to approach the aggressive resident mouse and clearly reduced their avoidance behaviour. SRA880 also selectively reduced aggressive behaviour in rats exposed to a ‘‘foreign’’ partner. These effects are produced in the range 0.1–30 mg/kg p.o.. SRA880 reduced contextual fear conditioning without affecting cue-elicited freezing in rats, whereas both parameters are reduced by classical anxiolytics. In mice, SRA880 improved the performance in step-down passive avoidance and enhanced retrieval-performance in step-through passive avoidance; SRA880 improved short-term memory in an object-recognition test. In rats, SRA880 specifically enhanced social recognition. These data show that an sst1 antagonist has profound central effects in models for neurological and psychiatric disorders. Support from EC Contract QLG3-CT-1999-00908 and Swiss grant BBW 00-0427. In vivo characterization of ACQ090, a somatostatin sst3 receptor antagonist D. Hoyer, A. Vassout, C. Gentsch, A. Enz, J.M. Vigouret, A. Jaton, P. Schoeffter, P. Kelly, A. Wrynn, K. Hurth, T. Troxler, Neuroscience Research, Novartis Institutes for BioMedical Research, WSJ 386/745. CH4002, Basel, Switzerland

ACQ090, [4-(3,4-Difluoro-phenyl)-piperazin-1-yl]{(4S,4aS,8aR)-2-[(S)-3-(6-methoxy-pyridin-3-yl)-2-methyl-propyl]-decahydro-isoquinolin-4-yl}-methanone, is an orally active, brain-penetrant somatostatin sst3 receptor antagonist. ACQ090 has high affinity for recombinant human and mouse somatostatin sst3 receptors (pKd = 8.15 and 8.31, respectively); it is devoid of intrinsic activity (pKb = 7.9). ACQ090 has >100-fold higher sst3 receptor affinity in comparison to other somatostatin receptors (except sst4 = 40 fold), monoamine or peptide receptors, ion channels or transporters. Behaviourally, ACQ090 (0.3–3 mg/kg p.o.) has marked sociotropic effects in intruder mice with increased social investigation towards the aggressive resident. ACQ090 (0.3–3 mg/kg p.o.) shows sociotropic effects in aggressive resident mice. ACQ090 (0.03–10 mg/ kg, p.o.) increases social exploration of an intruder towards a non-aggressive resident rat, similarly to chlordiazepoxide. ACQ090 (0.3–3 mg/kg p.o.) increases attention in the mouse intruder test, increases learning/ memory in the social recognition test similarly to oxiracetam, and increases the recognition index in the rat object recognition test, similarly to Exelon. ACQ090 (0.1–10 mg/kg p.o.) shows anxiolytic-like activity in stress-induced hyperthermia, but is inactive in the staircase test, suggesting that, in contrast to benzodiazepines, it might be more effective in social situations. Similarly to clinically effective antidepressants ACQ090 (10–30 mg/kg p.o.), given subchronically (but not acutely), has a pronounced effect in the modified rat forced swim test. Chronic ACQ090 (15–30 mg/kg p.o.) had a significant antidepressant-like effect in olfactory bulbectomised rats, similarly to tricyclics or SSRIs. These data show that an sst3 antagonist has profound central effects in models for psychiatric disorders. Support from EC Contract QLG3-CT-1999-00908 and Swiss grant BBW 00-0427. From VIP and PACAP to ADNP and NAP: New horizons in brain development and neuroprotection I. Gozes a, I. Pilzer a, R.A. Steingart a, D. Dangoor a, S. Rubinraut b, M. Fridkin b, DE Brenneman c, I. Divinski a, S. Furman a, a Clinical Biochemistry, Sackler Med. School, Tel Aviv Univ., Israel, b Organic Chemistry, Weizmann Inst., Rehovot, Israel; 3NICHD, Bethesda, MD, USA E-mail: [email protected] Background. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) share the same receptors and are implicated in neuronal survival. Inhibition of the expression of the splice variant of the receptor PAC1-HOP2 produced neuronal death in cell culture (1). Furthermore, knocking out activity-dependent neuroprotective protein

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(ADNP) that is upregulated upon incubation with VIP resulted in inhibition of brain formation and embryonic death in vivo and cell death in vitro. ADNP contains a short eight amino acid peptide sequence, NAP, which provides potent neuroprotection, in vitro and in vivo (2). Aims. To identify neuroprotective mechanisms, specifically, the receptor mediating VIP-neuroprotection; and downstream acting proteins. Methods: The PAC1 cDNA was PCR-cloned from rat cerebral astrocytes and genetically manipulated to obtain the HOP2 splice variant. It was then inserted into an expression vector and transfected into COS-7 cells that were used for 125I-VIP and 125I-PACAP binding assays. NAP interacting molecules were searched for using affinity chromatography with NAP conjugated to a solid support, followed by immunocytochemistry. Results. VIP and its agonist, Stearyl-Nle17VIP (SNV), bound to the cloned HOP2 PAC1 splice variant. The VIP antagonist Stearyl-Neurotensin6-11VIP728(SNH), that potently kills neurons, also bound HOP2. NAP was shown to bind tubulin, to promote microtubule polymerization and to protect against Zn2+ toxicity that is associated with microtubule re-organization. Further studies implied that ADNP interacts with microtubules. Conclusion. The activation of HOP2 participates in VIP/SNV-associated neuroprotection. ADNP and NAP that may act downstream to VIP to further provide cellular protection through interactions with the microtubular network that is essential for neuroglial function. Support: ISOA, Gildor Chair, BSF, Neufeld award, ISF, Allon Therapeutics.

References 1997. J. Mol. Neurosci. 9, 211. 2003. Dev. Brain Res. 144, 83.

Regulation and function of the Tachykinins K. Haddley, S. Vasiliou, E. Spencer, M. Howard, J.P. Quinn, Department of Physiology and Human Anatomy and Cell Biology, The University of Liverpool, UK E-mail: [email protected] Aim. To delineate the pathways that lead to the plasticity of neurotransmitter expression observed in anxiety, epilepsy, pain and axotomy. We have focused on tachykinin transmission and will determine which promoter domains of the substance P gene are targets for the cascade of signal transduction pathways that respond to various insults. We have more recently initiated a similar project on the NK1 Re-

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ceptor for Substance P. We will define both transcription factors and other intracellular modulators of gene expression. Methods and results. We have used reporter gene constructs for both promoters and addressed their ability to mimic endogenous gene responses. The reporter genes are delivered to neurons in vitro and in vivo as plasmids, virus vectors and transgenic constructs (Quinn et al., 2000). These constructs allow us to address function of transcriptional regulatory domains. However such domains involved in the tissue specific and stimulus inducible can be quite distant from the coding sequences. Bioinformatics can determine conserved regions flanking the coding sequence by comparison of the genomes of different species (MacKenzie and Quinn, 2004). Our hypothesis is that these conserved regions outside exons are involved in the expression of that gene. Conclusion. Major roles for the transcription factors Neuron restrictive silencer factor (NRSF) & bHLH factors (Quinn et al., 2002) in mediating gene expression to a variety of stimuli such as NGF, NOS and VIP has been determined. In the future combining the functional genomics (reporter genes) with bioinformatic information complemented by knowledge of functional polymorphisms that predispose to disease will create a picture of how gene expression is regulated dynamically.

References Quinn, J.P., Fiskerstrand, C.E., Gerrard, L., MacKenzie, A., Payne, C.M., 2000. Molecular models to analyse preprotachykinin-A expression and function. Neuropeptides 34, 292– 302. MacKenzie, A., Quinn, J.P., 2004. Post Genomic approaches to exploring neuropeptide gene mis-expression in disease. Neuropeptides 38, 1–15. Quinn, J.P., Bubb, V.J., Marshall-Jones, Z.V., Coulson, J.M., 2002. Neuron restrictive silencer factor as a modulator of neuropeptide gene expression. Regul Pept 108, 135–141.

Functional characteristics of peptide histidine-isoleucine (PHI) and peptide histidine-methionine (PHM) versus VIP and PACAP in vertebrate brain J.Z. Nowak a,b, A. Dejda a, J.B. Zawilska a,c, a Centre of Medical Biology and Microbiology, Polish Academy of Sciences, Lodz, Poland, b Department of Pharmacology, Medical University, Lodz, Poland, c Department of Pharmacodynamics, Medical University, Lodz, Poland E-mail: [email protected] Aim of investigation. PHI, PHM, PACAP and VIP are members of a superfamily of structurally related

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peptides, widely distributed throughout the body tissues, and displaying a pleiotropic biological activity. It is known that these peptides may act through common receptors, i.e., VPAC1 and VPAC2, whose main signaling pathways include AC-cAMP, and to a lesser extent PLC-IP3/DAG systems. In this work, we decided to compare the ability of PHI, PHM, PACAP38 and VIP to stimulate cAMP and IP3 formation, and to activate and translocate a Ca2+ and DAG-dependent protein kinase C (PKC) in the hypothalamus and cerebral cortex of chicks and rats. Methods. Experiments were carried out on slices of the hypothalamus and cerebral cortex isolated from white leghorn chicks (Gallus domesticus) and albino Wistar rats. The formation of [3H]cAMP was measured in slices prelabelled with [3H]adenine, while [3H]inositol phosphates ([3H]IPs) accumulation was analysed in slices prelabelled with myo-[3H]inositol. PKC activity was measured in membrane and cytosol preparation of the tested structures using [?-32P]ATP. Results. PACAP38 and VIP appeared to be a very potent stimulators of [3H]cAMP production in chick and rat cerebral cortex and hypothalamus; PHI and PHM were less potent in these systems, showing very weak activity in a chick model. PACAP38 and to a lesser degree PHI and PHM stimulated the accumulation of [3H]IPs in both tested models, being more potent in rat than in chick. In contrast, VIP showed a tendency to lower [3H]IPs accumulation. Both PACAP38 and VIP in a concentration-dependent manner increased PKC activity in membrane fraction and decreased the enzyme activity in cytosolic fraction of the rat and chick preparations. PHI and PHM only slightly affected the enzyme activity, especially in chick model. Conclusion. PACAP, VIP, and PHI/PHM may play a neuromodulator role in the vertebrate CNS. Depending on the species (mammals vs. avians) they can utilize different signaling pathways. PHI/PHM were generally weaker (than PACAP and VIP) stimulators of cAMP, IPs, or PKC. Acknowledgement. The study was supported by the Grant Nos. 2 P05A 097 26 and 3 P04C 076 25 from the State Committee for Scientific Research (KBN) in Poland.

Selected communications Co-chairs: Adolfo Aracil, Juana Gallar, Instituto de Neurociencias, UMH-CSIC, Sant Joan d’Alacant, Spain Urantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors V. Camarda a, W. Song b, E. Marzola c, M. Spagnol a, R. Guerrini c, S. Salvadori c, D. Regoli a, J.P. Thompson b,

D.J. Rowbotham b, S.A. Douglas d, D.J. Behm d, G. Calo’ a, D.G. Lambert b, a Pharmacology, University of Ferrara, Italy, b Department of Cardiovascular Sciences, University of Leicester, UK, cMed Chem. Univ. Ferrara, Italy, d GlaxoSmithKline, King of Prussia, PA, USA

Pharmacological profile of [Orn8]U-II on native and recombinant urotensin-II receptors V. Camarda a, R. Vergura a, R. Guerrini b, S.A. Douglas c, N. Aiyar c, H.M. Sarau c, M. Spagnol a, S. Salvadori b, D. Regoli a, G. Calo’ b, a Department of Pharmacology, University of Ferrara, Italy, b Department of Medicinal Chemistry, University of Ferrara, Italy, cGlaxoSmithKline, King of Prussia, PA, USA

Menthol induces neuropeptides release and neurogenic responses in different rodents species B. Campi a, R. Gatti a, A. Manni a, N. Lissi a, F. Benvenuti a, D. Gazzieri a, P. Geppetti b, S. Harrison a, M. Trevisani a, a Interdisciplinary Centre of Excellence for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy, b Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy

The antinociceptive effects of the opioid peptide endomorphin-1 are lost during chronic inflammation in rats Zongming Li, Jason J. McDougall, Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada T2N 4N1

Estrogen Modulation of DRG responses to nociceptive stimuli V. Chaban, Paul Micevych, Department of Neurobiology and Center for Neurovisceral Science and Women’s Health, University of California, Los Angeles, USA

GalR1, but not GalR2 or GalR3, is regulated by galanin signaling in the locus coeruleus through a cAMPdependent mechanism Jessica J. Hawes a, Darlene H. Brunzell, a David Wynick, c Venetia Zachariou a,b, Marina R. Picciotto a, a Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA, b U of Crete, Faculty of Med, Dept of Pharmacology, Crete, Greece. 3Bristol U, Dept of Medicine, Bristol BS2 8HW, UK

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Time-course expression of molecular mediators of TNBScolitis in mice: potential therapeutic effect of VIP C. Abad, M.G. Juarranz, A. Arranz, C. Martinez, M. Garcı´a, J. Leceta, R.P. Gomariz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain

PACAP controls sonic hedgehog functions in normal cerebellum and medulloblastoma: in vivo lessons from patched mutant mice Vincent LELIEVRE, Akop SEKSENIAN, Louise OZAKI, James WASCHEK, David Geffen School of Medecine, University of California at Los Angeles, USA

Urantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors V. Camarda a, W. Song b, E. Marzola c, M. Spagnol a, R. Guerrini c, S. Salvadori c, D. Regoli a, J.P. Thompson b, D.J. Rowbotham b, S.A. Douglas d, D.J. Behm d, G. Calo’ a, D.G. Lambert b, a Department of Pharmacology, University of Ferrara, Italy, b Department of Cardiovascular Sciences, University of Leicester, UK, c Department of Medicinal Chemistry, University of Ferrara, Italy, d GlaxoSmithKline, King of Prussia, PA, USA E-mail: [email protected] Aim of investigation. The recently identified urotensin-II (U-II) – UT receptor system regulates various biological functions especially at the cardiovascular level. The U-II related peptide [Pen5, dTrp7,Orn8]U-II(4-11) (urantide) has been recently proposed as a selective and potent UT receptor antagonist (Patacchini et al. (2003) In this study, we characterize the effects of urantide in the rat aorta bioassay and in a calcium mobilization assay performed on CHO cells stably expressing the human UT receptor (CHOhUT). Methods. Bioassay and CHOhUT calcium mobilization experiments were performed as previously described in detail (Camarda et al. 2002; Hirst et al. 1999). Results. In the rat aorta bioassay, urantide antagonized the contractile effects of U-II, behaving as a competitive (slope 0.94 ± 0.10 ), potent (pA2 8.24), and pure (no residual agonist activity up to 10 lM) antagonist. In the calcium mobilization assay, urantide produced a concentration dependent increase in [Ca2+]i (pEC50 8.11 ± 0.15; Emax 726 ± 147 nM), displaying slightly lower potency and maximal effects compare to U-II (pEC50 8.57 ± 0.27; Emax 917 ± 87 nM). Conclusion. The present results confirm pure antagonist behaviour of urantide in the rat aorta assay

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while revealing agonist activity of this UT receptor ligand in the CHOhUT [Ca2+]i mobilization assay. Although species-specific differences between rat and human UT receptor response to urantide cannot be excluded, we propose that urantide be classified as a low efficacy partial agonist. Differing efficiency of the stimulus-response coupling of the two assays, low for the rat aorta and very high for the CHOhUT [Ca2+]i, most likely influences the evaluation of urantide efficacy. References Patacchini, R.,Santicioli, P. Giuliani, S., Grieco, P., Novellino, E., Rovero, P., Maggi, C.A., et al., 2003. Urantide: an ultrapotent urotensin II antagonist peptide in the rat aorta. Br. J. Pharmacol. 140, 1155–1158. Camarda, V. Guerrini, R., Kostenis, E., Rizzi, A., Calo, G., Hattenberger, A., Zucchini, M., Salvadori, S., Regoli, D., 2002. A new ligand for the urotensin II receptor. Br. J. Pharmacol. 137, 311–314. Hirst, R.A., Harrison, C., Hirota, K., Lambert, D.G., 1999 Measurement of [Ca2+]i in whole cell suspensions using Fura-2. In: Calcium Signaling Protocols, Humana Press, Totowa, NJ, USA, pp.31–39 (Chapter 2).

Pharmacological profile of [Orn8]U-II on native and recombinant urotensin-II receptors V. Camarda a, R. Vergura a, R. Guerrini b, S.A. Douglas c, N. Aiyar c, H.M. Sarau c, M. Spagnol a, S. Salvadori b, D. Regoli a, G. Calo’ a, a Department of Pharmacology, University of Ferrara, Italy, b Department of Medicinal Chemistry, University of Ferrara, Italy, c GlaxoSmithKline, King of Prussia, PA, USA E-mail: [email protected] Aim of investigation. SAR studies performed on the cyclic portion of the peptide urotensin-II (U-II) revealed that the sequence Lys8–Trp7–Tyr9 is the most important for the U-II receptor (UT) occupation and activation. By substituting Lys8 with Orn8 we identified the novel UT ligand [Orn8]U-II whose detailed in vitro and in vivo pharmacological profile is presented in this study. Methods. Experiments were performed in vitro on HEK 293 cells expressing the human recombinant UT receptor (receptor binding, inositol phosphates (IPs) turnover, and calcium mobilization assays) and with the rat aorta bioassay and in vivo with the plasma extravasation assay in mice. Results. [Orn8]U-II binds human UT receptor with high affinity only sixfold lower than that of U-II (pKi 7.82 and 8.55, respectively). In HEK 293 cells, [Orn8]U-II induced IPs accumulation with potency and maximal effects lower than those of U-II (pEC50 8.85 and 9.40, Emax 224 ± 28% and 407 ± 25% over basal,

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respectively), while it behaved as full agonist with potency similar to that of U-II in the calcium assay (pEC50 9.36 and 9.41). In the rat aorta [Orn8]U-II antagonized in a competitive manner the contractile effects of U-II, with a pA2 value of 6.56, although showing some residual agonist activity. In in vivo studies, 30 nmol [Orn8]U-II antagonized plasma extravasation induced by 1 nmol U-II in several mouse tissues, without any statistically significant agonist effect. Conclusion. [Orn8]U-II behaved as UT receptor full agonist in the calcium assay, as a partial agonist in IPs turnover assay (? 0.5) and in the rat aorta bioassay (? 0.2), and as an antagonist in the mouse plasma extravasation assay. Thus, [Orn8]U-II should be considered as a UT receptor partial agonist whose estimation of efficacy strongly depends on the pharmacological assay employed. Acknowledgements. This study was supported by funds from UniFE (60% grants to S.S. and D.R.) and from the Italian Pharmacology Society (travel grant to C.V.).

Desensitization with capsaicin (10 lM) completely prevented the menthol induced CGRP release. Furthermore, menthol increased the basal level of intracellular calcium in cultured neurons, in a concentration dependent manner (EC50: 1.22 ± 0.29 lM). All the neurons that responded to menthol were also responsive to capsaicin. Finally, increasing concentration of menthol (0.1 lM–1 mM) induced a neurogenic contraction of guinea pig isolated bronchi (EC50: 101 ± 7 lM). Conclusions. The present data demonstrates that menthol, presumably through the activation of the TRPM8 receptor, induces different neurogenic responses in diverse rodents species. Furthermore, these results support the hypothesis of and keep open the question regarding the colocalization of the cold receptor and the hot receptor (TRPV1) on the membranes of the same sensory neurons. Acknowledgement. This work was supported by ICSI, University of Ferrara, Ferrara, Italy.

Reference McKemy et al., 2002. Nature. 416(7 March), 52–58.

Menthol induces neuropeptides release and neurogenic responses in different rodents species B. Campi a, R. Gatti a, A. Manni a, N. Lissi a, F. Benvenuti a, D. Gazzieri a, P. Geppetti b, S. Harrison a & M. Trevisani a, a Interdisciplinary Centre of Excellence for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy, b Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy E-mail: [email protected] Aim of investigation. The recently cloned ‘‘cold-receptor’’ (TRPM8) (McKemy et al., 2002) is an ion channel, belonging to the transient receptor potential (TRP) family. It senses temperatures between 8 and 28 C, and responds to cooling compounds, such as menthol and icilin. Activation of TRPM8 excites also a subpopulation of sensory neurons, that express the ‘capsaicin receptor’ (TRPV1). Here we have investigated whether menthol induces neuropeptide release from capsaicin-sensitive sensory neurons and causes neurogenic inflammatory responses. Methods. In this study we investigated the ability of menthol to induce: release of calcitonin gene-related peptide (CGRP) from the rat spinal cord; intracellular calcium mobilization in cultured rat trigeminal neurons; and neurogenic contraction of guinea pig isolated bronchi. Results. Menthol (1 lM) significantly increased the basal outflow of CGRP from slices of rat spinal cord (7.5 ± 1.3 fmol/g tissue, n = 6; P<0.05 vs. vehicle).

The antinociceptive effects of the opioid peptide endomorphin-1 are lost during chronic inflammation in rats Zongming Li, Jason J. McDougall, Department of Physiology and Biophysics, University of Calgary, Calgary, Canada, AB, T2N 4N1 E-mail: [email protected] Aim of investigation. Central administration of opioid drugs has been shown to modulate acute painful responses, however, the peripheral effects of endogenous opioid peptides in models of chronic inflammation are unknown. The current study explored whether peripheral administration of the endogenous -opioid neuropeptide endomorphin-1 to normal and chronically inflamed knee joints altered the ascending nociception signal generated in response to noxious rotation of the knee. Methods. In male Wistar rats (265–400 g), single unit extracellular recordings were made from knee joint fine afferent nerve fibres with a conduction velocity of 0.27– 12.0 m/s. Nerve activity was recorded in response to noxious hyper-rotation of the joint (25–40 mNm) both before and after close intraarterial injection of endomorphin-1 (10)11–10)8 mol). Comparisons were made between normal joints and chronically inflamed joints. Chronic inflammation was induced by intraarticular injection of Freund’s complete adjuvant 1 week prior to nerve recording.

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Results. In normal joints, endomorphin-1 caused up to 60% decrease in joint afferent activity following noxious hyper-rotation of the joint. This desensitising effect of the peptide was blocked by the selective -opioid receptor antagonist CTOP. Local administration of endomorphin-1 to adjuvant-inflamed joints had no significant effect on the action potential frequency evoked by noxious joint rotation. Conclusion. These data indicate that the antinociceptive effect of endomorphin-1 is attenuated during chronic inflammation probably due to downregulation and/or desensitization of articular -opioid receptors. This finding begins to explain the limited effectiveness of mu-opioid receptor agonists in alleviating chronic arthritic pain.

Estrogen modulation of DRG responses to nociceptive stimuli V. Chaban, Paul Micevych, Department of Neurobiology and Center for Neurovisceral Science and Women’s Health, University of California, Los Angeles, USA E-mail: [email protected] Background. One of the mechanisms of sex-related differences in pain perception underlying differences in clinical presentation of pain syndromes may be through the interaction of putative nociceptive stimuli and estrogen receptors (ER) on peripheral afferent nerve terminals. We have previously demonstrated that estrogen (17b-estradiol) rapidly attenuates the algesic, ATP-induced [Ca2+]i increase in cultured DRG neurons from rats1 and wild type (Wt) mice. In DRG neurons from ERa´KO mice, estradiol did not block ATP-induced [Ca2+]i flux indicating that the estrogen inhibition is dependent on ERa´2. Conversely, estrogen also attenuates anti-nociceptive cellular responses to -opioid receptors (MOR) agonists implying pro-nociceptive effects. Methods. Primary cultures of mice lumbosacral DRG neurons were collected under sterile technique, loaded with Fura-2 and tested for [Ca2+]i changes by fluorescent ratio imaging. A polyclonal antibody was used for ER and MOR immunocytochemistry. Results. Cultured DRG neurons expressed both ERa´ and ER and MOR by immunocytochemistry. ATP (20 M) and capsaicin (300 nM) an algesic agents, caused [Ca2+]i transients in small to medium size DRG neurons (20–35 M). A 5 min incubation with estradiol (100 nM) inhibited ATP-and capsaicin-induced [Ca2+]i. The effect of estradiol was reversible and receptor-mediated. Pretreatment with purinoreceptor antagonist PPADS, vanilloid receptor antagonist capsazepine or chelating extracellular Ca2+ with BAPTA (10 mM) eliminated [Ca2+]i transients. Estradiol coupled to bovine serum

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albumin (E-6-BSA), which does not penetrate the plasma membrane had the same effect, suggesting that estrogen acts at membrane-associated estrogen receptor. Conclusions. The present study demonstrates an important non-reproductive role of estrogen that can act as neuropeptides through membrane-associated ERa in modulating pro- and antinociceptive signaling.

References Chaban, V., Mayer, E., Ennes, H., Micevych, P., 2003. Estradiol inhibits ATP-induced [Ca2+]i increase in DRG neurons. Neuroscience 118 (6), 941–948. Chaban, V., Mayer, E., Ennes, H., Micevych, P., 2003. Rapid effects of 17b-estradiol on ATP-induced Ca2+ signaling in cultured dorsal root ganglion neurons from Wt and ERaKO mice. Gastroenterology 124 (4), A:1.

GalR1, but not GalR2 or GalR3, is regulated by galanin signaling in the locus coeruleus through a cAMPdependent mechanism Jessica J. Hawes a, Darlene H. Brunzell, a David Wynick, c Venetia Zachariou a,b, Marina R. Picciotto a, a Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA, b Department of Pharmacology, Faculty of Medicine, University of Crete, Crete, Greece, c Department of Medicine, Bristol University, Bristol BS2 8HW, UK E-mail: [email protected] Aim of investigation. Galanin attenuates opiate withdrawal symptoms and cAMP signaling cascades within the locus coeruleus (LC). Since GalR1 mRNA increases in the LC following opiate withdrawal, our aim was to elucidate the molecular mechanisms that regulate galanin receptor plasticity within the locus coeruleus (LC). Methods. GalR1, GalR2 and GalR3 were examined by western analysis following forskolin-stimulation of LC-like cells. Galanin binding and receptor immunoreactivity were examined in the LC of galanin wild type and knockout mice via [I125]-galanin equilibrium binding and immunohistochemistry, respectively. Results. GalR1, but not GalR2 or GalR3, is upregulated in the locus coeruleus (LC)-like Cath.a cell line in a cAMP-dependent manner. Upregulation of galanin binding in the LC of galanin knockout mice correlates with upregulation of GalR1, but not GalR2 and GalR3. Conclusion. GalR1 levels are regulated by neuronal activity and changes in cAMP levels. Since, the LC is critical for the onset and expression of autonomic and somatic symptoms of opiate withdrawal, these findings suggest that GalR1 signaling and neuroplasticity may

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play an important role in modulating signaling events underlying opiate addiction. Acknowledgement. This work was supported by the National Institutes of Health Grant No. DA15425. Reference Zachariou, V., Brunzell, D., Hawes, J., Stedman, D., Bartfai, T., Steiner, R., Wynick, D., Langel, U., M.R. Picciotto, 2003. The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal. Proc. Natl. Acad. Sci. USA. 100(15), 9028–9033.

Time-course expression of molecular mediators of TNBScolitis in mice: potential therapeutic effect of VIP C. Abad, M.G. Juarranz, A. Arranz, C. Martinez, M. Garcı´a, J. Leceta, R.P. Gomariz, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain E-mail: [email protected] Aim of investigation. Crohn’s disease is a chronic inflammatory pathology of the bowel that courses with abdominal pain and diarrhea, and causes considerable morbidity. Vasoactive intestinal peptide (VIP), a neuropeptide with immunoregulatory properties, has proved successful in the treatment of trinitrobenzene sulfonic acid (TNBS)-colitis in mice, an animal model that resembles human Crohn’s disease (Abad et al., 2003). Treatment with VIP reduced colitis and weight loss in mice, as well as mucosal damage and lymphocyte and macrophage infiltration in the colon of TNBS-animals. The aim of this study was to determine the molecular mechanisms of action of VIP on this model of colitis by analyzing the time-course expression of proinflammatory and regulatory mediators implicated in the pathophysiology of the disease. Methods. Colitis was induced by rectal instillation of 3 mg of TNBS in 50% ethanol, and mice were treated i.p. with VIP (1 nmol) on alternate days until sacrifice. Colons were removed on days 1, 3, 5 and 7 for RNA extraction, and samples were stored at -80 C until analysis. Real-time PCR analysis was performed to study mRNA levels of proinflammatory mediators, such as cyclooxygenase-2 (COX-2), IL-1?, IL-6, IL-12, IL-18, IFN?? and ?IL-4. Results. TNBS-treated animals showed an increased expression of all proinflammatory mediators studied, with a specific time course for each molecule. Thus, TNBS-mice showed high levels of of the enzyme COX-2, involved in the production of prostaglandins, that potenciate vascular permeability and cell migration to the inflammatory focus. Moreover, mice treated with TNBS showed elevated levels of the proinflammatory IL-1?, IL-12 and IL-18 cytokine mRNAs. Besides, mice ex-

hibited a Th1 response characterized by elevated levels of IFN? mRNA but low IL-4 in the chronic phase of the disease. This fact is correlated with the high expression of IL-12 and IL-18, that have been previously descibed as Th1 differentiating factors. Treatment with VIP decreases significatively the expression of all these mediators, showing an important antiinflammatory function of the immune response that explains the remission of the disease in the treated animals. VIP also plays a regulatory role of the lymphocytic response by favouring the Th2 versus Th1 response. Conclusion. The results show that VIP treatment ameliorates TNBS-colitis by inhibiting the expression of a wide range of proinflammatory molecules involved in the pathogenesis of the disease, supporting its role as a potential therapeutic agent for inflammatory diseases. Acknowledgement. This work was supported by the Spanish Department of Science and Technology, Grant No. BFI 2002-03489. Reference Abad, C., Martı´nez, C., Juarranz, M.G., Arranz, A., Leceta, J., Delgado, M., Gomariz, R.P., 2003. Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn’s disease. Gastroenterology. 124, 961–971.

PACAP controls sonic hedgehog functions in normal cerebellum and medulloblastoma: in vivo lessons from patched mutant mice Vincent Lelievre, Akop Seksenian, Louise Ozaki, James Waschek, David Geffen School of Medicine, University of California at Los Angeles, NPI-68-225, 740 Westwood Plaza, Los Angeles, CA 90095, USA E-mail: [email protected] Aims of investigation. Sonic hedgehog is one of the important patterning genes in ventral neural tube development. Loss of function elicited severe abnormalities of the developing brain, referred as holoprosencephaly in humans. Conversely, gene alterations leading to gain of function such as the disruption of the patched (ptc) gene, triggered patterning alteration and growth enhancement. Mice presenting a selective deletion of the ptc gene represents an elegant model to study shh-associated pathology. Indeed, ptc mutant mice present a lethal phenotype in embryos around E9– E10.5, while heterozygous animals develop normally in utero. However, these animals typically developed medulloblastoma and other tumor types, known in human as Gorlin syndrome. Methods. Because we recently demonstrated the ability of PACAP signaling to counteract shh proliferative action in external granular progenitor cells in mouse cerebellum, we decided to confirm and extend in vivo these findings, by monitoring the appearance of

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medulloblastoma ptc mutants with mice lacking the PACAP gene. Results. We herein report the successful breeding of mice that present a monoallelic disruption of the ptc gene with PACAP KO mice to generate double HZ mutants ptc/PACAP. More than 200 litters from these breedings have been obtained and observed for tumor development for over a 14-month period. Systematic genotyping first revealed low viability of ptc animals that completely lack PACAP. Second, the double heterozygous ptc/PACAP mutants presented exceptionally-high morbidity due to cerebellar tumors (medulloblastoma). We also reported the isolation and characterization of tumors cells isolated from mouse medulloblastomas. These cell lines displayed high levels of PAC1 receptor that are involved in repression of cell proliferation. Conclusions. These data represent the first in vivo demonstration of the potential control of shh actions by the neuropeptide PACAP and suggest putative therapeutic roles for PACAP in medulloblastoma treatment.

EtOH (0.001–3%) in the presence of the selective TRPV1 antagonist capsazepine (10 lM), the selective CGRP receptor antagonist CGRP-8.37 (10 lM) or their respective vehicles. Results. EtOH induced a concentration dependent relaxation (EC50: 1,17 ± 0,06; n = 20) in pre-contracted pig coronary artery. The maximum relaxation induced by EtOH (3%, Emax: 65 ± 5%, n = 20), expressed in % of maximum contraction of U46619 10 nM, was significantly attenuated by capsaicin pre-treatment (Emax: 28 ± 4%; n = 6, P < 0.05 vs. veh.), by capsazepine (Emax: 19 ± 12%; n = 6, P < 0.05 vs. veh.) and by CGRP8-37 (Emax: 12 ± 6%; n = 6, P < 0.05 vs. veh.). Conclusion. In this study we have demonstrated that EtOH relaxes small-size pig coronary arteries. This effect was mediated by a neurogenic mechanism that consists of CGRP release from sensory nerves via the activation of TRPV1. We propose that this novel mechanism contributes to the cardioprotective effect of the use of small doses of alcoholic beverages. Acknowledgement. Supported by ICSI, Ferrara, Italy.

POSTER SESSION 1

References

All authors in attendance on Monday 10th 12:00-13:00 h

Yuan-Jian, L., et al., 2002. Eur. J. Pharmacol. 442, 173–177. Trevisani, M., et al., 2002. Nat. Neurosci. 5, 546.

Ethanol (EtOH) relaxes isolated pig coronary arteries via a CGRP and TRPV1 dependent mechanism D. Gazzieri a, T. Carrara a, N. Ardizzoni a, M. Trevisani a, I. Angeli b, F. Tarantini b, P. Geppetti b, S. Harrison a, a Interdisciplinary Centre of Excellence for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy, b Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy E-mail: [email protected] Introduction and aims. Capsaicin-sensitive sensory nerves are known to innervate the heart. In addition, calcitonin gene related peptide (CGRP), released from these nerves, protects the heart against ischemic damage. Recently, we demonstrated that ethanol (EtOH) stimulates primary sensory neurons by the activation of the vanilloid receptor-1 (TRPV1), thus causing the release of peptides such as CGRP and substance P. Our aim was to study the effect of EtOH in isolated pig coronary artery in relation to TRPV1 activation. Methods: Secondary coronary artery (diameter of 3 mm) of the pig was removed, suspended in Krebs solution (aerated 95% O2 and 5% CO2 at 37 oC) and allowed to equilibrate for 60 min (tension of 1.8 g). Cumulative concentration response curves were performed, in pre-contracted (U46619, 10 nM) tissues, with

Noxious heat induces neuropeptides release from central endings of nociceptors by a TRPV1-indipendent mechanism A.C. Manni a, N. Lissi a, B. Campi a, R. Gatti a, A. Girardi a, S. Harrison a, P. Geppetti, b, M. Trevisani a, a Interdisciplinary Centre of Excellence for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy, b Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy E-mail: [email protected] Background. The Transient receptor potential vanilloid 1 (TRPV1) belongs to the TRP family of cation channels. TRPV1 is activated by different stimuli (osmotic/mechanical stress, endogenous mediators, noxious heat). Although noxious heat (43–51 C) is an adequate stimulus for TRPV1 activation in neuronal cell bodies, no data are available on central terminals of capsaicinsensitive primary sensory neurons. Aim and methods. The aim of the present study was to examine the involvement of TRPV1 on neuropeptides release induced by noxious heat (48 C) from central terminals of capsaicin-sensitive primary sensory neurons. Results. Noxious heat induced a significant increase in CGRP outflow from slices of rat spinal cord (26.3 ± 2.9 fmol/g tissue, n = 8, P < 0.05 vs. control).

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Pretreatment with the TRPV1 antagonist, capsazepine (10 lM) did not produce any significant reduction in the CGRP release (26.5 ± 8.3 fmol/g tissue, n = 8, vs. vehicle). Similarly, no inhibitions were observed by using other two TRPV1 antagonists: SB 366791 (10 M) and iodo-resiniferatoxin (I-RTX, 100 nM), 27.3 ± 6.3 fmol/g tissue, n = 4 and 18.5 ± 5 fmol/g tissue, n = 4, respectively. Discussion and Conclusion. In the present study we have shown that noxious heat induces CGRP release from central terminals of primary sensory neurons. Different TRPV1 antagonists (capsazepine, SB366791 and I-RTX) did not inhibit the CGRP release from rat spinal cord. In conclusion, in contrast with cell bodies, excitation of central terminals of capsaicin-sensitive sensory neurons by noxious heat results from a TRPV1independent mechanism. This work was supported by Interdisciplinary Centre of Excellence for the Study of Inflammation, University of Ferrara, Italy.

lM). Furthermore, H2S increased CGRP release from slices of rat spinal cord in a concentration dependent manner (3 mM, 20.5 ± 6.1 fmol/g tissue, n = 6; P < 0.05 vs. vehicle). Desensitisation by pre-treatment with capsaicin (10 lM) completely inhibited CGRP outflow induced by H2S. Conclusion. The present data demonstrate the ability of H2S to excite cultured capsaicin-sensitive primary sensory neurons and to induce the release of sensory neuropeptides from their nerve endings. Acknowledgement. This work was supported by ICSI, University of Ferrara, Italy. References Goodwin, L.R., Francom, D., Dieken, F.P., Taylor, J.D., Warenycia, M.W., Reiffenstein, R.J., Dowling, G., 1989. J. Anal. Toxicol. 13(2) March–April,105–109. Patacchini, R., Santicioli, P., Giuliani, S., Maggi, C.A., 2004. Br. J. Pharmacol. March 29.

Hydrogen sulphide (H2S) induces calcitonin gene-related peptide (CGRP) release and intracellular calcium mobilization in primary sensory neurons N. Lissi a, A.C. Manni. a, B. Campi a, A. Girardi a, R. Gatti a, D. Gazzieri a, L. Tarsitano a, P. Geppetti b, M. Trevisani a, S. Harrison a, a Interdisciplinary Centre of Excellence for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy, b Department of Critical Care Medicine and Surgery, University of Florence, Florence, Italy E-mail: [email protected]

Modulatory role of capsaicin-sensitive sensory neurones on oxazolone-induced allergic contact dermatitis in mouse ear ´ . Ba´nvo¨lgyi a, T. Berki b, L. Pa´linka´s b, Zs. E. Pinte´r a, A a Helyes , A. Grant c, J. Szolcsa´nyi a, S.D. Brain c, a Department of Pharmacology, Faculty of Medicine, University of Pe´cs, Hungary, b Department of Immunology, Faculty of Medicine, University of Pe´cs, Hungary, c Centre for Cardiovascular Biology and Medicine, King’s College London, Guy’s Campus, SE1 1UL, UK E-mail: [email protected]

Background. Hydrogen sulphide (H2S) is a colourless gas that smells like rotten eggs (from the sulphur) and it is extremely toxic. H2S acts as an irritant to eyes, nose, throat and lungs, and as an internal poison causing paralysis of the respiratory system and unconsciousness. H2S is also present in the brain and it may exhibit neurotransmitter-like functions (Goodwin et al., 1989). Furthermore, acute exposure to H2S causes inflammation, hyperpnoea–apnoea, and death. Although the mechanism of these effects have to be clarify, it has recently been reported that H2S may stimulate terminals of primary sensory neurons (Patacchini et al., 2004). Aim and Methods. In the present study, we have evaluated the ability of H2S to induce the release of calcitonin gene-related peptide (CGRP) from rat spinal cord and to increase the intracellular calcium level in cultured rat trigeminal neurons. Results. H2S significantly increased the intracellular calcium level in cultured trigeminal neurons (89.5 ± 18.13 nM). All the neurons that responded to H2S were capsaicin sensitive. The H2S -mediated effects were significantly reduced by pre-treatment with capsaicin (10

Aim of investigation. It is supposed that pro-inflammatory and anti-inflammatory sensory neuropeptides released from capsaicin-sensitive nerve endings modulate the immune responses. The aim of the present work was to learn more about this function in oxazolone-induced allergic contact dermatitis model in the ear of BALB/c, and three transgenic mice where NK1 or TRPV1 receptor or CGRP genes were knocked out. Methods. Mice (20–30 g) were anaesthetised with ketamine or isoflurane. Sensitisation was induced by 2% oxazolone to the shaved abdomen on two consecutive days and hypersensitivity was elicited 6 days later by 2% oxazolone on both sides of the right ear. The left ear was treated with 96% ethanol. Ear swelling was measured by an engineer’s micrometer over 72 h. Mice were killed and ears were cut for the immunological assays and histology. The number of accumulated neutrophils was determined by spectrophotometric measurement of myeloperoxidase enzyme. The cytokine profile was characterized by cytometric bead array (CBA) using a kit containing Th1/Th2 antibodies (IL-2, IL-4, IL-5, TNF-a, IFN-c). Resiniferatoxin (RTX) pretreatment

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was used to impair the function of capsaicin-sensitive sensory nerves. In order to examine the role of NK1 receptors, an antagonist SR140333 (240 nmol/kg) was administered before oxazolone. Results. Oxazolone induced 65–80% increase of ear thickness after 24–48 h. Ear oedema was more enhanced after depletion of sensory neuropeptides by RTX and in TRPV1 receptor knockout animals. Pretreatment with SR140333 and the absence of NK1 receptors or CGRP inhibited the ear swelling. Accumulation of myeloperoxidase positive cells was not significantly affected by the used manipulations. Prominent increase of IL-4, TNF- a´ and IFN- level was detected in the ear samples to prove that these cytokines play key role in the oxazolone-induced contact allergy. The cytokine profile was influenced by sensory neuropeptides. Conclusion. The results suggest that NK1 receptors and CGRP play a pro-inflammatory role in ear oedema. Conversely responses in RTX-pretreated and TRPV1 knockout mice propose a protective role of neurogenic components mediated via the TRPV1 receptor. Modulatory role of sensory neuropeptides has not been shown on neutrophil accumulation. We conclude that sensory neurones may influence the contact hypersensitivity reaction via pro- as well as anti-inflammatory mechanisms. Acknowledgement. Supported by The Wellcome Trust International Development Award, and Hungarian Research Grant No. OTKA T-032548. E.P. and Zs.H. held Janos Bolyai Fellowship.

Does the vanilloid receptor (VR1) mediate anandamideinduced vasodilatation in the rat knee joint? W.S. Luk, F.Y. Lam, Department Pharmacology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China E-mail: [email protected] Aim of investigation. Anandamide is an endocannabinoid that has agonist activities on cannabinoid receptors (CB1 and CB2) and the vanilloid receptor (VR1). The vasodilator action of anandamide is thought to involve VR1-mediated release of neuropeptides from sensory neurones. This hypothesis was tested on the vasodilator action of anandamide in the rat knee joint. Methods. A laser Doppler perfusion imager was used to measure changes in knee joint blood flow in the anaesthetized rat. Anandamide was administered topically onto the exposed knee joint, and various receptor antagonists were tested on its effect to elucidate the receptor type(s) involved. Results. Anandamide (10 nmol) produced 32% increase in the knee joint blood flow. This effect was substantially reduced in the presence of the vanilloid receptor antagonist capsazepine (5 nmol), the tachykinin NK1 receptor antagonist SR140333 (10 nmol), the

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CGRP1 receptor antagonist CGRP8-37, or the anandamide transporter inhibitor AM404. On the other hand, the cannabinoid CB1 receptor antagonist AM281 and the cannabinoid CB2 receptor antagonist AM630 produced only minor inhibition on the response. Conclusion. The vasodilator action of anandamide in the rat knee joint involves activation of VR1 and the release of vasodilator neuropeptides, which could be substance P and calcitonin gene-related peptide. Cannabinoid receptors do not play a significant role in mediating this effect. Acknowledgement. A Direct Grant for Research and a postgraduate studentship to Miss WS Luk were provided by The Chinese University of Hong Kong for this work.

Activity of NK1 agonists and antagonists on Gassignaling using a cAMP-responsive luciferase gene reporter E. Perdona’, F. Emms, M. Corsi, M.J. Garnier, Department of Biology, GlaxoSmithKline S.p.A., Psychiatry Centre of Excellence for Drug Discovery, Verona, Italy E-mail: elisabetta.2.perdona’@gsk.com Aim of investigation. The neurokinin receptor NK1 is coupled to both the aq and as G-protein subunits in vitro. Whereas the pharmacological characterisation of SPA and endogenous ligands of NK1 receptors on Gaqmediated signaling has been well documented, little is known about these ligands’ activity on the Gas -mediated NK1 activity (Host et al., 2001). The present study was aimed at further characterizing the Gas-mediated activity of a series of NK1 receptor agonists and antagonists previously characterised at NK1-mediated Gaq signaling. Methods. Gas-mediated signaling was assessed using a cAMP-responsive luciferase gene reporter transiently expressed in chinese hamster ovary cells stably expressing the human NK1 receptor. Results. Substance P (SP) and Neurokinin A (NKA) were potent NK1 receptor agonists increasing luciferase expression in a dose-dependent manner with distinct profiles. NKA (EC50 = 0.62 ± 0.06 nM, n = 6) was sixfold less potent than SP (EC50 = 0.10 ± 0.01 nM, n = 6) and exhibited a partial agonist profile producing a maximal stimulation of 68 ± 6% relative to SP. The activity of the two NK1 receptor antagonists L-733,060 and GR205171 was further evaluated in this assay. L733,060 (100 and 300 nM) produced a rightward shift of both SP- and NKA-mediated increase in luciferase expression without affecting agonist efficacy. Analysis of the mean curves from 3 independent experiments showed that 100 nM L-733,060 had similar potency at antagonising SP and NKA with apparent pKb values of

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8.6 and 8.8, respectively. To the contrary, GR205171 (0.3–3 nM) antagonised SP and NKA stimulatory activities on luciferase expression in a non-surmountable manner. The reduction of both SP and NKA maximal responses was of about 20% in presence of 0.3 nM GR205171. Conclusion. These data suggest that both Gaq- and Gas-mediated signaling pathways might play a role in triggering NK1 receptor mediated biological effects.

Conclusion. NGF regulation of the proximal promoter in both types of neurons requires the BHLH site 60 to mediate the increase in expression observed. Further we have shown that in an in vitro model of seizure that KA application upregulates PPTA expression and that this may be mediated in part, by the binding of bHLH factors to the -60 Ebox. Acknowledgement. Funded by the Merseyside regional epilepsy association and the BBSRC.

Reference

Deciphering the role of the tachykinin pathway in pain, anxiety and depression by the generation of a "humanised" mouse model for the NK1 receptor S. Vasiliou, J. Quinn, University of Liverpool, Liverpool, UK E-mail: [email protected]

Holst, B., Hastrup, H., Raffetseder, U., Martini, L., Schwartz, T.W., 2001. Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusion and mutagenesis. J. Biol. Chem. 276, 19793–19799.

Regulation of the preprotachykininA/substance P gene promoter in the central (CNS) and peripheral nervous system (PNS) in rodents. A role for basic helix–loop–helix factors K. Haddley, L. Gerrard, J. Quinn, Physiology Department, University of Liverpool, UK E-mail: [email protected] Aim. Inappropriate regulation of SP is a major factor in the development of allodynia and hyperalgesia in the PNS. Additionally, following seizure SP is markedly upregulated in the dentate gyrus and pyramidal layers of the rat hippocampus in the CNS. We addressed the role of the proximal rat preprotachykinin A (PPTA) promoter to mediate some of these changes in expression and compare mechanisms of regulation in both models. Methods. Adeno-associated virus containing a 1kb fragment of rat PPTA proximal promoter ± a previously characterised basic helix–loop–helix (bHLH) protein binding site (E box element at -60) supporting the luciferase marker gene were used to infect rat dorsal root ganglion (DRG) cell cultures. Plasmids containing the same constructs and those expressing bHLH transcription factors were nucleofected into rat hippocampal cell cultures. Stimuli were applied and luciferase expression assayed. Results. In an in vitro model of pain/inflammation in adult rats, NGF (100 ng/ml) upregulates the PPTA promoter. Mutation of the -60 Ebox abolished the upregulation of the PPTA promoter in response to NGF in DRG cultures. Similarly in neonates, the mutation of the -60 Ebox resulted in a 2–4 fourfold increase in promoter activity compared to the wild type construct in DRG cultures. In an in vitro model of seizure, in neonatal hippocampal cell cultures, 2-4 fold upregulation of the PPTA promoter in response to kainic acid (5 mM) or NGF is abolished by mutation at the -60 Ebox or by co-expression of a dominant negative bHLH factor.

Aim. The tachykinin pathway has been implicated in the regulation of diverse behavioural manifestations such as pain, anxiety, depression, stress responses and reward behaviours. Current preclinical rodent models appear to be inappropriate for the study of this pathway, due to the differential expression of the tachykinins and/or their receptors in rodent and human and also inter-species differences in amino acid structures of these proteins that render them inappropriate targets for pharmaceutical intervention using drugs which are going into clinical trials We have attempted to overcome some of these obstacles by inserting the human genes Pre-protachykinin-A (encoding the neuropeptides Substance P and NKA) and the tachykinin receptor NK1 into the mouse genome. Methods. A yeast artificial chromosome (YAC) containing the entire human NK1 receptor locus and upstream regulatory elements (377 kb) was manipulated by the incorporation of the marker gene green fluorescence protein (GFP), which would allow visualisation of transgene expression. This YAC construct was expected to comprise all the sequence information required for transgene expression that would mimic the endogenous gene expression patterns. The transgenic construct was microinjected in vitro into mouse oocyte pronuclei. Results: We have generated three transgenic lines for the human NK1 receptor. Expression of the human NK1 gene has been observed in coronal sections of the brain and spinal cord by GFP visualisation. The site of incorporation of the transgene and the transgene copy number are currently being investigated by fluorescent in situ hybridisation (FISH) and Southern blotting. Conclusion. The ability to manipulate yeast artificial chromosomes by homologous recombination and introduce human genes into animal models, will allow us to address questions regarding human gene regulation and function. Crossing the PPT-A and NK1 human alleles onto the relevant ablated genetic backgrounds

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(PPT-A and NK1 )/) already available), we are aiming to construct an animal that only expresses the human genes. Such a ‘‘humanised" animal model will be a valuable tool for preclinical pharmacological and behavioural studies. Further with increased awareness of genetic polymorphisms predisposing to disease we will be able to address the function of human specific polymorphic domains in situ. Acknowledgements. This work was funded by the BBSRC and MSD.

The regulation and function of the tachykinin in vitro and in vivo models of epilepsy E. Spencer, J. Quinn, Physiology Department, University of Liverpool, Liverpool, UK E-mail: [email protected] Background. Modulation of neuropeptide expression after status epilepticus is observed with neuropeptides that are either pro or anti-convulsant in nature. In vivo studies have shown that there is a strong correlation between the incidences of epilepsy, augmented levels of substance P (proconvulsant), reduced levels of galanin (anti-convulsant) and the integrity of the dentate gyrus in rodent models. Modulation of neuropeptide expression will result from changes in the active transcription factor complement in the cell in response to the epilepsy trigger. The transcription factor neuronal restrictive silencing factor (NRSF/REST) is a key candidate in regulation of neuropeptide gene expression during epilepsy based on our studies in other models. At least two NRSF isoforms are induced in response to seizure in rodents and correlated with modulation of the PPT-A gene expression. NRSF is thought to be a repressor of neuronal gene expression whereas the REST 4 isoform encodes a truncated protein, which has potential to antagonise the full length NRSF resulting in anti-silencer function. Alterations in REST expression are thought to account for the changes in differential neuropeptide expression in hippocampal regions of kainic acid (KA) induced seizure models. Results. Organotypic cultures of rat hippocampal sections were used as an in vitro model to look at changes in PPT, Galanin, REST and REST 4 gene expression over time post KA stimulation. RT-PCR analysis showed that in control conditions fluctuations of PPT and Galanin expression occur in opposition in response to serum. However KA stimulation overrides this response, causing these genes to escape their normal control mechanisms and probably be deregulated. RTPCR and western blotting has confirmed the presence of REST 4 expression in the in vitro organotypic slice model. Preliminary immunostaining of epileptic rat brains indicated that REST 4 expression is located in the cytoplasm of hippocampal degenerative neurons.

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Conclusions and future work. Preliminary findings indicate that organotypic slice model may be used to reflect the mechanisms of gene expression, which occur in vivo during epilepsy. To draw further conclusions of neuronal gene plasticity, real time PCR will be carried out on the in vitro model complemented with western blotting analysis. Self sustained status epilepticus will be induced by the perforant path model in vivo in a series of tachykinin transgenic mice. Temporal and spatial topology of the neuropeptides and transcription factors will be compared between the transgenic mice and with in vitro studies. We hope in this manner to delineate signal transduction pathways that result in the longterm differences to gene plasticity associated with the recurrence of seizure rather than the immediate mechanisms that result in the initial seizure. Acknowledgements. Alison Roberts and Anja Kipar for the immunostaining. This work was funded by The Wellcome Trust.

Role of NK1 tachykinin receptors in the development and resolution of cerebral oedema in rat R. Vink a, C. Howard a, M. Ghabriel a, A. Zacest a, P. Blumbergs a, A. Nimmo b, a Department of Pathology, University of Adelaide, Adelaide, b Department of Biomedical Sciences, James Cook University, Townsville, Australia E-mail: [email protected] Aim of Investigation. Cerebral oedema is a major clinical problem. In younger victims of traumatic brain injury (TBI), oedema is responsible for 50% of all deaths. We have shown that neurogenic inflammation plays a role in the development of cerebral oedema (Nimmo et al., 2004). The present study examines the role of substance P in the development of cerebral oedema, and the potential for NK1 antagonists to resolve it. Methods. TBI was induced in rats using the weight drop model. The NK1 antagonist, N-acetyl tryptophan (NAT; 1 mol/kg), was administered i.v. 30 min after injury. Cerebral oedema was assessed by magnetic resonance imaging (MRI). Changes in substance P- and aquaporin-4-immunoreactivity (IR) in brain were examined following trauma by immunohistochemistry. Results. The substance P-IR associated with cortical blood vessels increased significantly within 5 h of injury. MR imaging demonstrated the concurrent development of profound cerebral oedema. In animals receiving postinjury administration of NAT, there was marked resolution of this oedema. Levels of aquaporin-4-IR associated with cortical blood vessels were significantly reduced following injury. However, in animals treated with NAT, the aquaporin-4 levels were greater than pre-injury.

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Conclusion. Substance P appears to play a role in cerebral oedema, whilst NK1 antagonists may resolve it. Following trauma, there is an increase in substance P levels associated with cortical blood vessels and parallel development of severe oedema. This oedema is markedly resolved following post-injury administration of NAT. These effects would appear to be related to changes in the expression of aquaporin-4. Following injury, there is a loss of aquaporin-4 staining around cortical vessels. This effect was reversed by administration of NAT. In conclusion, NK1 antagonists may provide a therapeutic intervention for managing cerebral oedema. Acknowledgement. Supported by the NH & MRC, Australia.

Reference Nimmo, A., et al., 2004. Neurogenic inflammation is associated with the development of edema following TBI in rats. Neuropeptides 38, 40–47.

The role of NK1 receptors in vanilloid-induced emesis and genital grooming in Suncus murinus (house musk shrew) J.A. Rudd, M.K. Wai, C. Wan, Department of Pharmacology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong E-mail: [email protected] Aim of investigation. Previous studies have shown that resiniferatoxin (RTX) induces emesis and genital grooming in Suncus murinus that may be mediated by neuropeptide release (Rudd and Wai, 2001; Andrews et al., 2000). The present studies extend these observations to an investigation of other pungent and nonpungent vanilloids. The potential role of NK1 receptors in the RTX-induced behaviours was also investigated using CP-122,721. Methods. Drugs were injected via a previously implanted cannula into the lateral ventricle of male Suncus murinus. The vanilloids used in the study were RTX, capsaicin, olvanil, and phorbol 12-phenylacetate 13acetate 20-homovanillate (PPAHV). The animals were observed for 90 min for episodes of emesis and/or genital grooming. Results. RTX (0.1–3 nmol), capsaicin (30–100 nmol), and PPAHV (10 nmol) induced emesis with maximum episodes of 11.0 + 1.3, 6.0 + 1.0, and 5.7 + 1.9, respectively (P < 0.05). Olvanil (10–600 nmol) displayed a non-significant trend to induce emesis (P > 0.05). Only RTX (1–10 nmol) was capable of eliciting genital grooming; the maximum response was 46.7 + 8.8 episodes at 1 nmol (P < 0.05). A 5 min pretreatment with CP-122,721 (100–300 nmol, i.c.v.) antagonized the emesis (maximum reduc-

tion was approximately 77%; P<0.05) but not the grooming (P > 0.05) induced by RTX (10 nmol). Conclusion. With the exception of olvanil, all of the vanilloids tested in this study were found to be emetic. RTX was the only agent capable of eliciting genital grooming. NK1 receptors may participate in the mechanism of RTX-induced emesis but not in RTX-induced genital grooming. Acknowledgement. Partly supported by the RGC of Hong Kong (CUHK2000.1.060).

References Rudd, J.A., Wai, M.K., 2001. Genital grooming and emesis induced by vanilloids in Suncus murinus, the house musk shrew. Eur. J. Pharmacol. 422, 185–195. Andrews, P.L.R., Okada, F., Woods, A.J., et al., 2000. The emetic and anti-emetic effects of the capsaicin analogue resiniferatoxin in Suncus murinus, the house musk shrew. Br. J. Pharmacol. 130 1247–1254.

Increase in activity of rat spinal trigeminal neurons during both slow administration of a nitric oxide donor and hypocapnia – involvement of sensory neuropeptides? S.V. Koulchitsky, P.I. Terekhin, M.J.M. Fischer, K. Messlinger, Institute of Physiology and Experimental Pathophysiology, University of Erlangen-Nu¨rnberg, Germany E-mail: [email protected] Aim of investigation. Clinical observations have shown that nitric oxide (NO) donors as well as calcitonin gene-related peptide (CGRP) can provoke headache attacks particularly in persons suffering from primary headaches. It was also demonstrated that these headache attacks were accompanied by an increase in CGRP in the venous outflow from the head (Juhasz et al., 2003) and altered cerebrovascular regulation. Our aim was to investigate changes in central trigeminal neuronal activity during slow continuous infusion of the NO donor sodium nitroprusside (SNP) or artificial hyperventilation (HV) in an animal model of meningeal nociception. Methods. In thiopental anaesthetised rats extracellular action potentials were recorded from spinal trigeminal neurones that received afferent input from the exposed parietal dura mater. Vital parameters were monitored. SNP (50 lg/kg) was infused continuously i.v. over 2 h. HV was induced by increasing respiratory rate and the extent thereof was standardized by a fall in ETCO2 from 4.5 ± 0.3% to 2.5 ±x0.4%. Results. Infusion of SNP was accompanied by a slowly developing and continuous increase in the spontaneous firing rate of trigeminal neurones. HV was accompanied by significant increases in the spontaneous firing rate of STN neurones followed by a slow recovery

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to the baseline level after the end of the challenge. No significant change in mean systemic arterial blood pressure was observed during all experiments. Conclusion. Our group has previously shown that NO stimulates the release of CGRP from rat dura mater (Strecker et al., 2002). Based on these data and the present study we suggest that a continuous release of pro-nociceptive neuropeptides such as CGRP may be involved in the increase of spontaneous neuronal activity provoked by NO. A change in carbon dioxide concentration and pH due to HV may cause an increase in endogenous NO production. These hypotheses will now be tested using neuropeptide receptor antagonists. Acknowledgements. The study was supported by the BMBF and the Alexander von Humboldt-Foundation. References Juhasz, G., et al., 2003. Pain 106, 461–470. Strecker, T., et al., 2002 J. Vasc. Res. 39, 489–496.

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flammatory substance (SP, PGE2 or BK, always as 10–4 M, 0.2 ml) resulted in a reversible decrease of the movement evoked activity compared with such activity measured before (25%, 9% and 12%, respectively). Conclusion. We conclude that NOS inhibitors influence the sensitization to movement evoked activity of fine afferents in the acutely inflamed knee joint. The effects of NOS inhibitors on sensitizing effects were significant for SP (p < 0.05) but not for BK or PGE2. The results show that NO takes part in the modulation of activity of peripheral fine articular afferents. However, the mechanism of action (e.g. direct or indirect) and the participation of the different NOS remains to be elucidated. Acknowledgment. This project was supported by the Deutsche Forschungsgemeinschaft, SFB 353/B10 and a grant of the Max-von-Frey Gesellschaft.

Effects of nitric oxide synthase inhibitors on the mechanosensitivity of articular nociceptors of inflamed knee joints in rats M. Pawlak, R.F. Schmidt, Physiologisches Institut der Universitt, Wu¨rzburg, Germany E-mail: matthias. [email protected]

UFP-101, a potent and pure antagonist selective for the nociceptin/orphanin FQ receptor – summary of in vitro and in vivo findings G. Calo’ a, A. Rizzi a, R. Guerrini b, S. Salvadori b, D.G. Lambert c, D. Regoli a, a Department of Pharmacology, University of Ferrara, Italy, b Department of Medicinal Chemistry, University of Ferrara, Italy, b Department of Cardiovascular Sciences, University of Leicester, UK E-mail: [email protected]

Aim of investigation. Tissue injury and inflammation result in the release of proinflammatory mediators, some of which cause pain and hyperalgesia. Furthermore nitric oxide (NO) is also involved in the modulation of the release of proinflammatory neuropeptides and can contribute to the induction and maintenance of sensitization processes. The aim of the present study was to examine the effects of NO synthase (NOS) on the action of proinflammatory factors in the inflamed knee joint of the rat. Methods. An acute inflammation was induced using kaolin and carrageenan. Single afferent fibres innervating the knee joint were isolated from the saphenous nerve and electrophysiologically characterized. Mechanical stimulation consisted in controlled inward and outward rotations of the knee joint within and outside its normal working range. Each of the four movements lasted 10 s and a movement cycle was started every 3 min. Knee joint afferent mechanosensitivity was tested for every inflammatory agent used (substance P, SP; prostaglandin E2, PGE2 and bradykinin, BK) before and after close intraarterial bolus application. The effect of nitric oxide synthase (NOS) inhibitors were tested by subsequent i.a. applications of L-NAME or 7-NINA close to the knee joint. Results. Application of NOS inhibitors (10–3 M, 0.2 ml) immediately after the application of a proin-

Aim of investigation. Nociceptin/orphanin FQ (N/ OFQ) modulates several biological functions by activating a specific G-protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we summarised the in vitro and in vivo data obtained in the last two years with the NOP ligand, [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101, (Calo et al., 2002). Methods. Experiments were performed in vitro with biochemical, bioassay, neurochemical, and electrophysiological techniques, and in vivo in animal models of pain, locomotion, mood, and cardiovascular and renal functions. Results. UFP-101 binds with high affinity (pKi 10.2) and selectivity (3000-folds over opioid receptors) the hNOP receptor and antagonizes N/OFQ cellular effects in different functional assays (cAMP, GTPgammaS binding, isolated tissues, release of NA and 5-HT from synaptosomes; K+ current in slices from different brain areas; chemotaxis in human monocytes) with high potency (pA2 7–8). In vivo, UFP-101 antagonises N/OFQ: (i) pronociceptive and antinociceptive effects in the tail withdrawal assay, (ii) inhibitory effects in the mouse locomotor activity assay, (iii) reduction of HR, BP and NA blood levels in guinea pigs; and (iv) inhibitory effects on mesolimbic dopamine release. Moreover, per se, UFP-101 produces after icv administration: (i) a small

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but long lasting anti-nociceptive effect; (ii) a potentiation of stress induced analgesia, (iii) antidepressant-like effects in the forced swimming; and (iv) conditioned place preference. Conclusion. Collectively, these findings demonstrated that UFP-101 behaves, in different in vitro and in vivo assay, as a potent, and selective NOP receptor antagonist. Moreover these data suggest that NOP receptor antagonists are worthy of further evaluation as mild analgesics and especially as anti-depressant agents.

101 (pA2 6.9–7.3) but not to naloxone, and no longer present in tissues (vas deferens and synaptosomes) taken from mice lacking the NOP receptor gene. Preparation/assayN/OFQUFP-102concentration ratio CHOhNOP GTPg[35S]8. 7010. 1226CHOhNOP cAMP9. 8610 172mVD7. 769. 4448mC8. 909.2 12RVD7. 248. 5721gpI7. 949. 2420m Synaptosomes 8. 649. 6210. Conclusion. Overall we conclude that UFP-102 behaves as a highly potent and selective NOP receptor ligand and to date represents the most potent full agonist described in literature.

Reference Calo’, G., Rizzi, A., Rizzi, D., Bigoni, R., Guerrini, R., Marzola, G., Marti, M., McDonald, J., Morari, M., Lambert, D.G., Salvadori, S., Regoli, D., 2002. [Nphe1,Arg14,Lys15]N/OFQ-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br. J. Pharmacol. 136, 303–311.

UFP-102, a novel and potent peptide agonist for the nociceptin/orphanin FQ receptor – in vitro studies A. Rizzi a, G. Carr a, F. Mela a, R. Guerrini b, J. McDonald c, D.G. Lambert a, S. Salvadori b, D. Regoli a, G. Calo’ a, a Department of Pharmacology, University of Ferrara, Italy, b Department of Pharmaceutical Science, University of Ferrara, Italy, c Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, UK E-mail: [email protected] Aim of investigation. Nociceptin/orphanin FQ (N/ OFQ) and its receptor (NOP) are implicated in a variety of physiological processes both at central and peripheral levels. The identification of selective ligands is required to determine the potential of the NOP receptor as a target of innovative drugs. [(pF)Phe4,Arg14,Lys15]N/ OFQ-NH2 (UFP-102) has been recently generated in our laboratories and here we describe the in vitro pharmacological characterisation of this novel NOP ligand. Methods. Human recombinant NOP receptors expressed in Chinese hamster ovary cells (CHOhNOP) and native animal receptors expressed in isolated tissues were used. Binding studies and functional assay in CHOhNOP, the mouse/rat vas-deferens (m/rVD), mouse colon (mC) and guinea pig ileum (gpI), and [3H]5-HT overflow from mouse cerebral cortex synaptosomes were investigated as previously described (Calo et al., 2002). Results. UFP-102 bound with high affinity to the hNOP (pKi 11.32) showing at least 300-fold selectivity over classical opioid receptors. In functional studies UFP-102 mimicked the effects of N/OFQ showing similar maximal effects but higher potencies (see table) These effects of UFP-102 were sensitive to the NOP selective antagonists J-113397 (pA2 7.7–8.1) and UFP-

Reference Calo, G., Rizzi, A., Rizzi, D., Bigoni, R., Guerrini, R., Marzola, G., Marti, M., McDonald, J., Morari, M., Lambert, D.G., Salvadori, S., Regoli, D:, 2002. [Nphe1,Arg14,Lys15]N/OFQ-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br. J. Pharmacol. 136, 303–311.

UFP-102, a novel and potent peptide agonist for the nociceptin/orphanin FQ receptor - in vivo studies A. Rizzi a, G. Marzola a, R. Guerrini b, S. Zucchini a, V.A. Kenigs c, D.R. Kapusta c, E.C. Gavioli a, S. Salvadori b, D. Regoli a, G. Calo’ a, a Department of Pharmacology, University of Ferrara, Italy, b Department of Pharmaceutical Science, University of Ferrara, Italy, c Department. of Pharmacology, LSU, Health Sci. Ctr., New Orleans, LA, USA E-mail: [email protected] Aim of investigation. [(pF)Phe4,Arg14,Lys15]N/OFQNH2 (UFP-102) is a novel peptide ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). UFP-102 has been generated by combining, in the N/ OFQ-NH2, sequence two chemical modifications ([Arg14,Lys15] and [(pF)Phe4]) which have been previously demonstrated to increase N/OFQ potency. Methods. The effects of supraspinal (i.c.v.) and spinal (i.t.) UFP-102 administration were studied in the tail withdrawal assay (TW) in Swiss and C57/BL6J-129 NOP+/+ and NOP)/) mice (Calo et al., 1998); while the effects of UFP-102 on cardiovascular and renal functions were evaluated in conscious rats according to Kapusta et al. (1997). Results. In the TW assay, UFP-102 (0.01–1 nmol) produced pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t., mimicking the actions of N/OFQ. However, UFP-102 was at least 10-fold more potent than N/OFQ and produced longer lasting effects. The pronociceptive and antinociceptive effects of UFP-102 were prevented by the selective NOP receptor antagonist UFP-101 and absent in NOP)/) animals. In rats, like N/OFQ (5 nmol, i.c.v.), UFP-102 (0.05 nmol, i.c.v.) produced a marked and

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sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. Conclusion. Collectively, these findings demonstrate that UFP-102 behaves, in different in vivo models, as a potent and selective NOP receptor agonist which produces long lasting effects. References Calo, G., Rizzi, A., Marzola, G., Guerrini, R., Salvadori, S., Beani, L., Regoli, D., Bianchi, C., 1998. Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay. Br. J. Pharmacol. 125. 373–378. Kapusta, D.R., Sezen, S.F., Chang, J.K., Lippton, H., Kenigs, V.A., 1997. Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin /orphanin FQ. Life Sci. 60, PL15-21.

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cific norbinaltorphimine showed only slight blocking effects compared with naloxone. The results obtained in the in vitro functional assays are in good agreement with the opioid agonist effect seen in the in vivo pain test. Conclusion. Incorporation of d-Ala2 and d-Nle5 into MERF resulted in improved analgesic potency and changed opioid receptor selectivity of the ligand. These properties of DADN, together with its higher metabolic stability and availability in radiolabeled form, make it a very promising tool for studying the function of the opioid receptors. Acknowledgement. This work was supported by OTKA T025711 and NKFP 027/2001.

Pharmacological and functional studies of d-Ala2-d-Nle5enkephalin-Arg-Phe (DADN) F. To´th a, G. Horva´th b, M. Szikszay b, J. Farkas a,G. To´th a, A. Borsodi a, S. Benyhe a, a Institute of Biochemistry, BRC, HAS, Szeged, Hungary, b Deparment of Physiotherapy, University of Szeged, Szeged, Hungary E-mail: [email protected]

A biological study on newly synthesized DTPA chelating group containing nociceptin derivative ¨ . Gu¨ndu¨z a, M. Ligeti b, A. Magyar b, E. Kato´ c, A. O Ro´nai c, C. Vita d, F. Hudecz b, A. Borsodi a, S. Benyhe a, a Institute of Biochemistry, Biological Research Center, HAS, Szeged, Hungary, b Research Group of Peptide Chemistry, Eo¨tvo¨s L. University, HAS, Budapest, Hungary, c Group of Neuropsyhopharmacology, Semmelweis Medical University, HAS, Budapest, Hungary, d Departement d’Ingenierie et d’Etudes des Proteines, Centre D’Etudes de Saclay, Saclay, France E-mail: [email protected]

Aim of investigation. Several synthetic derivatives of MERF (Tyr-Gly-Gly-Phe-Met-Arg-Phe) were investigated to gain structure-activity relationship information and to develop novel analogues with improved stability. One compound in this series, containing double d-amino acid replacements at the 2nd and 5th position, Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) has been shown to change the receptor type selectivity / compared to MERF. DADN has been radiolabeled and radioreceptor binding assays confirmed the -receptor selectivity of the ligand. In the present work a broader characterization of the peptide is provided. Methods. (i) direct binding assays on CHO cells expressing cloned opioid receptors. (ii) functional approach ([35S]GTPS assay). (iii) in vivo pharmacological experiments (tail-withdrawal test). Results. In the radioligand binding assays the highest affinity for [3H]DADN binding was observed in CHO cells transfected with l-opioid receptors, confirming the l-selectivity of the peptide. The peptide produced dose-dependent increases in [35S]GTPS binding and the stimulation was inhibited by l-receptor specific antagonists further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat tail-withdrawal tests. Among the selective opioid antagonists tested, the -selective naltrindole and the -spe-

Aim of investigation. Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the ORL1 (Opioid Receptor Like 1) receptor system which is involved in pain regulation, tolerance and dependence to opioids as well as many other physiological and pathophysiological events. Radiopharmaceutics are used for imaging or therapy. DTPA (diethylenetriaminepentaacetic acid) is a commonly used chelator in radiopharmaceuticals. DTPA was incorporated into the [Arg14,Lys15]nociceptinNH21 at the e-amino group of Lys15. Two DTPA containing peptides were isolated following the peptide synthesis and purification. According to the mass spectrometric analysis the products are: [Arg14,Lys(DTPA)15]nociceptin-NH2 and its cross linked dimer. These derivatives were further investigated in receptor binding and functional biochemical assays. Our further aim is to prepare radiolabeled nociceptin derivative with a selective and high affinity binding to ORL1 receptor. Methods. Peptide synthesis was carried out by solid phase synthesis, biological properties of these compounds are studied in rat brain membranes by: radioligand binding, functional biochemical GTPS binding assays and mouse vas deferens (MVD) bioassay. Results. Increasing concentrations of newly developed nociceptin derivatives competed for the nociceptin binding sites. Higher affinities (0.78 ± 0.1 and 5.94 ± 1.9 in nM) than nociceptin were observed. These

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compounds stimulated G-protein binding in a dose-dependent manner similar to nociceptin (up to 182.1 ± 4% and 255.7 ± 11.3% of basal). Conclusions. Nociceptin derivatives containing DTPA displayed not only similar in vitro binding characteristics to nociceptin but also more potent agonistic properties were obtained from the bioassays. These compounds can be radiolabelled and further used diagnostically and therapeutically.

Reference Okada, K. et al., 2000. BBRC 278, 493–498.

POSTER SESSION 2 All authors in attendance on Tuesday 11th 12:00–13:00 h

Analgesic effect of TT-232, a heptapeptide somatostatin analogue, in acute pain models of the rat and the mouse and in streptozotocin-induced diabetic hyperalgesia ´ Szabo´ a, E. Pinte´r a, G. Zs. Helyes, a, K. Bo¨lcskei a, A a a Peth , K. Elekes , R. Alma´si a, R. Bo¨rzsei a, T. Szts b, Gy. Ke´ri c, J. Szolcsa´nyi a,d, a Department of Pharmacology Pharmacotherapy, Pe´cs, Hungary, b Biostatin Research Development Ltd., Budapest, Hungary, c Peptide-biochem Research Group The Hungarian Academic of Science, Department of Medical Chemistry, Semmelweis University, Budapest, Hungary, d Neuropharmacology Research Group The Hungary Acade-mic Science, Pe´cs, Hungary E-mail: [email protected] Aim of investigation. Somatostatin released from capsaicin-sensitive sensory nerves exerts systemic antiinflammatory and antinociceptive actions. TT-232 is a stable, peripherally-acting heptapeptide somatostatin analogue with a highest affinity for sst4 receptors. It has been shown to inhibit acute and chronic inflammatory responses and sensory neuropeptide release from capsaicin-sensitive nociceptors. In the present study the antinociceptive effects of TT-232 were analysed using both acute and chronic models of nociception. Methods. For examining acute somatic chemonociception, 100 ?l 2.5% formalin was injected s.c. into the left paw of male Wistar rats and composite pain score (CPS) was calculated in the early (0–5 min) and late phases (25–45 min). Noxious heat threshold and resiniferatoxin (RTX, 0.05 nmol i.pl.)-evoked thermal allodynia was measured on increasing temperature hot plate. Diabetic neuropathic mechanical hyperalgesia was tested in rats 6 weeks after i.v. injection of 50 mg/kg streptozotocin. Phenylquinone (0.02%, 0.2 ml i.p.)-

evoked abdominal contractions were examined in Balb/c mice. Results. TT-232 (80 lg/kg i.p.) significantly inhibited CPS in both phases of the formalin test. The minimum dose to elevate the noxious heat threshold and diminish the heat threshold drop evoked by resiniferatoxin (RTX 0.05 nmol i.pl.) was 20 and 10 lg/kg i.p., respectively, as measured by an increasing-temperature hot plate. TT232 (10–200 lg/kg s.c.) significantly inhibited writhing movements in mice, but within this dose range no clear dose-response correlation was found. Five weeks after streptozotocin administration (50 mg/kg i.v.) the diabetes-induced decrease in the mechanonociceptive threshold was inhibited by 10–100 lg/kg i.p. TT-232. Conclusion. These findings show that TT-232 potently inhibits acute chemical somatic/visceral and thermal nociception and diminishes chronic mechanical hyperalgesia associated with diabetic neuropathic condition, thereby it could open new perspectives in the treatment of various pain syndromes. Acknowledgement. Helyes Zs, Pinte´r E, Peth G were supported by J. Bolyai Fellowships.

Somatostatin immunoreactive neurons and fibers in the brain of the urodele amphibian Pleurodeles waltl A. Gonza´lez, N. Moreno, R. Morona, M. Mun˜oz, J.M. Lo´pez Department of Cell Biology, Faculty of Biology, University of Complutense, Madrid, Spain E-mail: [email protected] Aim of investigation. To get insights into the evolution of the somatin(SOM)-containig neuron systems in vertebrates we accomplished a detailed study on the distribution SOM immunoreactivity in the brain of Pleurodeles waltl. An additional goal of our study on the SOM system in the urodele brain was to determine the localization of SOM in catecholaminergic and nitrergic neurons. Methods. Antibodies against mammalian S14 appears a good tool for studying SOM-like immunoreactivity in the brain of this urodele, since S14 is the most constant form of SOM through phylogeny. Combination of SOM immunohistofluorescence with that of tyrosine hydroxylase (TH) or nitric oxide synthase (NOS) was used. Results. Intense immunoreactivity was observed in neurons and fibers throughout the brain. Within the telencephalon, the subpallial regions were densely labeled containing both cells and fibers, primarily in the striatum and amygdala. The majority of the neurons were located in the preoptic area and hypothalamus, and less numerous cells were also found in the thalamus. A conspicuous innervation of the median eminence was revealed, which arise from the hypothalamic cell populations. In the brainstem, intense fiber labeling was present in the tectum and tegmentum, whereas cell

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bodies were located only in the tegmentum of the mesencephalon and in the interpeduncular, raphe and reticular nuclei of the rhombencephalon. Longitudinal fiber tracts throughout the brainstem were observed and they continued into the spinal cord in the laterodorsal funiculus. TH and SOM only colocalized in a cell population in the ventral preoptic area. In turn, the striatum and amygdala contained neurons with SOM and NOS. Conclusion. The SOM neuronal system in the brain of P. waltl shares many features with those of amniotes. Colocalization of SOM with catecholamines and nitric oxide is very restricted in the urodele brain, but in places that can be easily compared to those reported for mammals, suggesting that interactions between these neurotransmitter systems are a primitive feature shared by tetrapod vertebrates. Acknowledgement. Supported by MCYT, BFI200303756.

A novel ligand with unique functional properties at the human somatostatin receptor subtype 4 M. Engstro¨m, J. Tomperi, K. El-Darwish, M.J. hman, J.M. Savola, S. Wurster, Juvantia Pharma Ltd, Turku, Finland E-mail: [email protected] Aim of investigation. Our aim was to identify new non-peptide compounds selective for the human somatostatin receptor subtype 4 (h sst4) and to characterize these compounds for their functional activities in vitro. Methods and Results. We discovered a novel compound, coded J-2156, for which competition binding assays indicated a nanomolar affinity on the h sst4 and over 400-fold selectivity against the other four somatostatin receptor subtypes. We found J-2156 to elicit an agonist response on the h sst4 about three times as efficacious as those of the two endogenous ligands, somatostatin-14 (SRIF14) and somatostatin-28 (SRIF-28), when evaluated via a membrane-based [35S]guanosine-5-O-(3-thio)triphosphate binding assay. That the endogenous peptides are clearly less efficacious than J-2156 could also be verified by demonstrating that SRIF-14 displays the typical behavior of a partial agonist. That is, increasing concentrations of SRIF-14 caused a concentration dependent rightward-shift of the dose-response curves for J-2156 without affecting the maximal response. Further, in a whole-cell functional assay (Cytosensor Microphysiometry), we found the responses towards SRIF-14 and SRIF-28, but not that to J-2156, to be susceptible to agonist-induced desensitization of the h sst4.

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Conclusion. We conclude that the endogenous ligands SRIF-14 and SRIF-28 do not define the maximal extent of agonism achievable on the h sst4 and that J-2156 represents a so-called ‘superagonist at this receptor. The finding that J-2156, despite its high efficacy, shows a much lower propensity to cause desensitization of h sst4-mediated responses than SRIF-14 and SRIF-28 is unique, since empirically strong agonists have been observed to induce more, not less desensitization of receptor-mediated responses than weak agonists. The properties of superagonism and a low propensity for causing desensitization allow for a much more pronounced and sustained stimulation of h sst4 than previously possible. Acknowledgement. Supported in part by the National Technology Agency of Finland (Tekes).

Different receptors mediate the vascular actions of somatostatin and octreotide in rat knee joints F. Lam, E. Ng, Department of Pharmacology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China E-mail: [email protected] Aim of investigation. Somatostatin is considered as an anti-inflammatory neuropeptide, but recently we have shown that it has pro-inflammatory actions in the rat knee joint (Lee and Ng, 2003). In this study, we have compared the actions of somatostatin and its analogue octreotide on blood vessels of the rat knee joint. Methods. Somatostatin and octreotide were tested on blood flow and plasma extravasation in the rat knee joint, measured by laser Doppler perfusion imaging and Evans blue extravasation, respectively. Results. Topical administrations of somatostatin and octreotide onto the exposed rat knees produced similar dose-dependent increases in blood flow that peaked at 10 nmol. Intra-articular injections of 10 nmol of the two agonists also elicited comparable plasma extravasation. The somatostatin receptor antagonist cyclo-somatostatin (40 nmol) did not affect the somatostatin-induced responses, but it almost abolished those produced by octreotide. Conclusion. Somatostatin and octreotide are vasodilators and inducers of plasma extravasation in the rat knee joint. These pro-inflammatory actions of octreotide are mediated by somatostatin receptors that can be blocked by cyclo-somatostatin. However, similar actions produced by somatostatin are mediated by a different population of somatostatin receptors, which is resistant to inhibition by cyclo-somatostatin.

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Acknowledgement. This research was supported by the Direct Grant for Research of The Chinese University of Hong Kong. Reference Lam F, Ng E., 2003. Characterisation of somatostatin actions on knee joint blood vessels of the rat. Eur J Pharmacol 474, 295–301.

Effect of VIP treatment in the cytokine balance in NOD mice F. Rosignoli, V. Roca, J. Leceta, Perez-Leiros, R.P. Gomariz, Departamento de.Quı´mica Biolo´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires-CONICET, Argentina, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain E-mail: [email protected] Aim of investigation. The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type 1 diabetes and autoimmune sialadenitis. NOD mice develop a Th1 spontaneous response against exocrine glands. Vasoactive intestinal peptide (VIP) is a common messenger to immune and nervous systems that modulates innate and adaptative immunity (Gomariz et al., 2001).VIP has recently emerged as a promising candidate for treatment of inflammatory Th1-driven diseases as rheumathoid arthritis and Crohn’s disease. We have reported a lower activity of nitric oxide synthase and salivary flow as an early alteration in salivary glands of prediabetic NOD mice that precedes the onset of the cellular autoimmune response (Rosignoli and Pe´rez Leiro´s, 2002). Here we investigated for the first time the potential of VIP treatment in an early stage to modulate the release of cytokines involved in inflammatory and Th1 responses in NOD mice. Methods. NOD mice (10 weeks old) were treated with VIP (1 nmol) as a single dose at every other day for 2 months. Then we measured the levels of TNFa´, IFN and IL-10 in serum, supernatants of stimulated splenocytes (anti-CD3, anti-CD28) and in homogenates of salivary glands and pancreas. The levels of IL-12 were measured in serum. Results. Treatment with VIP in NOD mice reduced the levels of TNFa in serum, spleen cell supernatants and homogenates of pancreas and submandibular glands, but did not modify them in homogenates of parotid glands. Regarding IL-10 we could only observe an increase in submandibular glands with no changes in the other tissues. The levels of IL-12 diminished in serum of NOD mice treated with VIP respect to control NOD mice and IFNg only decreased in splenocytes of VIP-injected mice.

Conclusion. Prediabetic NOD mice VIP treatment at early stages, modulates the expression of inflammatory/ Th1 cytokines both at systemic and target organs level. Our study demonstrates, for the first time the possible therapeutic effect of VIP in a spontaneous model of Th1 autoimmune disease. Acknowledgement. Rosignoli’s fellowship was supported by Fundacio´n Antorchas from Argentina. This work was supported in part by the Spanish Department of Science and Technology Grant BFI 2002-03489. References Gomariz, R.P., Martinez, C., Abad, C., Leceta, J., Delgado, M., 2001. Immunology of VIP: a review and therapeutical perspectives. Curr. Pharma. Design 7, 89–111. Rosignoli, F., Pe´rez Leiro´s, C:, 2002. Nitric oxide synthase I and VIPactivated signaling are affected in salivary glands of NOD mice. J. Neuroimmunol. 130, 109–16.

VIP inhibits collagen-induced arthritis by regulating NFB and AP-1-dependent transcription M.G. Juarranz, C. Abad, A. Arranz, Garcı´a M, C. Martinez, R.P. Gomariz, J. Leceta, Departamento de Biologı´a Celular, Facultad de Biologı´a, Universidad Complutense, Madrid, Spain E-mail: [email protected] Aim of Investigation. Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology that leads to chronic inflammation in the joints and subsequent destruction of the cartilage and bone. TNFa´ has a central role in joint inflammation, since it induces a cascade of pro-inflammatory factors including IL-1, IL-6, IL-12 or nitric oxide as well as several pro-inflammatory chemokines. Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, modulates both the innate and adaptive immunity, showing a predominant anti-inflammatory action. Recently, we have described that VIP significantly reduces incidence and severity of arthritis in an experimental model in mice, completely abrogating joint swelling and destruction of cartilage and bone (Delgado et al., 2001), as well as, in humans by reducing the production of proinflammatory cytokines (Martinez et al., 2004). The therapeutic effect of VIP was associated with downregulation of both inflammatory and autoimmune components of the disease. This study examines the role of VIP in the modulation in synovial cells of two important transcription factors involved in the disease, NF-B and AP-1. Methods. Male DBA/1J mice (6–10 wk of age) were injected intradermally at the base of the tail with 0.15 ml of emulsion containing 200 g CII and then boosted i.p. with 200 lg CII in PBS 21 days after the primary immunization. 1 nmol of VIP was given intraperitoneally

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per mouse every other day between day 30 and 40 after first immunization. Then nuclear and cytoplasmic extract of synovial cells were obtained, and aliquots were stored at –80C for later use in EMSAs and immunoblotting studies. Results. VIP was able to decrease NF-B nuclear translocation and its subsequent DNA binding with an increase of IKB protein levels in the cytoplasm of these synovial cells. Although VIP did not significantly affect AP-1 binding in synovial cells, it modified its complex composition. Whereas in control arthritis mice, AP-1 complex in synovial cells is composed by c-Jun and cFos homo and heterodimers. VIP treatment substituted c-Jun presence in the AP-1 complex by JunB (a negative component of the complex). This effect is correlated by a VIP-mediated decrease in c-Jun phosphorylation. This effect on transcription factors is correlated by the fact that synovial cells from VIP treated mice showed decreased production of the inflammatory cytokines IL-6, TNFa´ or IL-1 and chemokines like CXCL1/MIP-2a or CCL2/MCP-1. By contrast VIP-treated synovial cells showed an increased production of the anti-inflammatory cytokine IL-10. Conclusion. Our data show that VIP treatment ameliorates RA by modulating NF- kB and AP-1, two transcriptional factors involved in RA. This results confirm VIP as an alternative candidate for the development of treatments for RA. Acknowledgement. This work was supported by the Spanish Department of Science and Technology Grant BFI 2002-03489. References Delgado, M., Abad, C., Martinez, C., Leceta, J., Gomariz, R.P:, 2001. Vasoactive intestinal peptide prevents experimental arthritis by down-regulating both autoimmune and inflammatory components of the disease. Nat. Med. 7, 563–68. Juarranz, M.G., Santiago, B., Torroba, M., Gutierrez-Can˜as, I., Palao, G., Galindo, M., Abad, C., Martinez, C., Leceta. J., Pablos, J.L., Gomariz, R.P., 2004 Vasoactive intestinal peptide modulates proinflammatory mediator sı´nthesis in osteoarthritic and rheumatoid synovial cells. Rheumatology 43, 416–22.

VIP prevents experimental multiple sclerosis by downregulating both inflammatory and autoimmune components of the disease A. Fernandez-Martin a, E. Gonzalez-Rey a, J. Martin a, D. Ganea b, M. Delgado a, a Institute of Parasitology and Biomedicine, CSIC, Granada, Spain, b Department of. Biological Sciences, Rutgers University, Newark, NJ E-mail: [email protected] Aim of Investigation. Multiple sclerosis (MS) is a chronic inflammatory autoimmune neurodegenerative disease causing important neurological disorders. MS is characterized by two pathologic components: in-

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flammation of CNS and CD4 Th1-mediated autoimmunity, which lead to an autoimmune attack against myelin in the CNS. Therefore, the actual pathologic strategies treat to decrease or to prevent both components of the disease. The vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that functionally has various characteristics to be considered as a possible therapeutic agent for MS. Firstly, VIP is a potent anti-inflammatory agent, at systemic level as well as at the CNS level, which regulates a broad spectrum of proinflammatory factors synthesized by both activated macrophages and microglia. Secondly, following antigenic stimulation, VIP is able to shift a Th1 immune response (which is deleterious for MS) to a Th2 response (which is beneficial for MS pathology). The therapeutic effect of VIP on a model of rheumatoid arthritis and Crohn’s disease, two inflammatory and Th1-mediated autoimmune diseases, demonstrates its efficacy to treat this kind of disorders. Methods. We investigated the therapeutic effect of VIP in the principal animal model for the study of MS, experimental autoimmune encephalomyelitis (EAE), with which shares many immune, histopathologic and clinical features. After different profiles of administration, we determined VIP effect on clinical score, disease incidence, histopathology, as well as cellular and molecular mechanisms involved. Results. Treatment of EAE mice with VIP on alternate days following disease onset prevented and ameliorated, in a dose-dependent manner, the clinical score and incidence of the disease. From a histopathologic point of view, VIP treatment diminished the infiltration of inflammatory cells in the brain and spinal cord parenchyma and the subsequent demyelinization characteristic of EAE. VIP treatment prevents inflammation in CNS parenchyma: VIP-treated mice showed decreased expression of proinflammatory mediators (cytokines, chemokines, inflammatory enzymes) in CNS parenchyma in comparison with untreated EAE mice. VIP treatment prevents autoimmune reaction: Lymph node T cells from VIP treated mice are much less autoreactive than those control EAE mice, and showed a Th2-like profile versus the Th1-like profile of EAE mice. This was correlated with the fact that in contrast to EAE controls, adoptive injection of lymph node T cells from VIP-treated mice was unable to transfer the disease to health hosts. In addition, VIP-treated EAE mice showed lower levels of autoantibodies against myelin components. Conclusions. In this study, by using the EAE model, we showed VIP as a very attractive candidate to treat MS. VIP prevents and ameliorates the incidence and deleterious consequences of the disease by downregulating its two pathologic components: inflammation and autoimmunity.

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VIP induces the generation of regulatory T cells. A new therapy for autoimmunity and transplantation? E. Gonzalez-Rey a, A. Fernandez-Martin a, E. Gonzalez-Rey a, D. Ganea b, M. Delgado a, a Institute of Parasitology and Biomedicine, CSIC, Granada, Spain, b Department of Biological Sciences, Rutgers University, Newark, NJ, USA E-mail: [email protected] Aim of Investigation. Autoimmune diseases, a serious health problem in the world, are thought to result from breaks in tolerance, brought upon by abnormalities in the recognition of exogenous or endogenous antigens. Regulatory T cells (Treg) has been recently found to be essential in maintaining tolerance against self-antigens, and in re-establishing immune homeostasis following a full-blown immune response. The fast developing field of Treg remains quite confusing, and although two major CD4+ subsets can be distinguished based on the mechanism for suppression, very little is known about the immune factors inducing Treg generation/activation. The controlled development and/or activation of antigen-specific Treg is of obvious relevance for autoimmune diseases and transplantation. Vasoactive intestinal peptide (VIP), a peptide produced by the immune system, acts as an immunomodulatory factor regulating both innate and adaptive immunity, showing a predominant anti-inflammatory action, resulting in important therapeutic effects on various autoimmune diseases. Here we propose that VIP induces the generation and/or activation of Treg, which then play an essential role in implementing the VIP anti-inflammatory functions. We investigated the generation and/or activation of Treg by VIP in vivo, characterized the VIP-induced Treg in terms of phenotype, antigenspecificity, and mechanisms for suppression, and extend our investigation into several models of autoimmunity (multiple sclerosis and arthritis) and of allogeneic transplantation, evaluating the role of antigen-specific Treg in the protective effect of VIP. Results. Injection of VIP in a antigen-specific TCRtransgenic mouse in the presence of low doses (tolerogenic) of antigen resulted in an increased percentage of cells with the Treg phenotype (CD25+, CD45RBlow, CD69+, CD62L+, Foxp3+, CD134hi) in both draining lymph nodes and spleen. These cells were functional Treg, measured by its suppressor activity, because in cocultures with antigen-stimulated CD4 T cells, they inhibited their proliferation. VIP-induced Treg in vivo produced high levels of TGFb and IL-10, and expressed high amounts of CTLA4, all of them mediators of Treg function. In fact, VIP-induced Treg cells exerted their suppressive action mainly through a CTLA4-cell contact-dependent manner, and partially through a TGFb/ IL10-soluble mediators-dependent manner. In vivo VIP induced Treg maintain their suppressive activity upon

transfer to a different host, as demonstrated by using a classical in vivo method for suppressive activity, i.e., inhibition of delayed type hypersensitivity (DTH). Finally, of obvious therapeutic application is the fact that VIP-induced Treg were able to prevent the adoptive transfer of two autoimmune diseases, such as multiple sclerosis (using the EAE model) and rheumatoid arthritis (using the CIA model), and to increase the survival in a model of allogeneic transplantation of bone marrow cells. Conclusions. Immunomodulatory peptides, such as VIP, could be used for the design of in vitro systems for the generation of antigen-specific regulatory T cells, which will open new therapeutic avenues in transplantation and autoimmunity.

A neural basis for the loss of vasoactive intestinal peptideinduced vasodilatation in acutely inflamed rat knee joints A. Ku¨rsat Barin & Jason J. McDougall, Department of Physiology & Biophysics, University of Calgary, Calgary, AB, T2N 4N1 Canada E-mail: [email protected] Aim of Investigation. Vasoactive intestinal peptide (VIP) is known to act on vascular smooth muscle where it causes potent vasodilatation. VIP has also been found to activate rat connective tissue mast cells (MC), leading to the release of inflammatory mediators. This study examined the vasomotor effect of VIP in normal and acutely inflamed knee joints. The contribution of MCs to this vasomotor response was also investigated. Methods. Blood flow in normal and acutely inflamed male Wistar rat knee joint capsules was assessed by laser Doppler imaging. Acute inflammation was induced by intraarticular injection of 2% kaolin/2% carrageenan, followed by a 3 h recovery period prior to experimentation. VIP (10–13–10–9 mol) was topically applied to exposed knee joints and the percentage change in perfusion from control was determined. Synovial MC contribution to the VIP response was studied by prior stabilization of the MCs with cromolyn (20 mg/kg; topical). To assess the role of joint nerves on the vasomotor effects of VIP in acutely inflamed rats, surgical disruption of the nerves supplying these joints was performed in eight animals 1 wk prior to induction of inflammation and perfusion measurement. Results. VIP (10–13–10–9 mol) dose-dependently increased synovial perfusion (P < 0.0001, one-way ANOVA, n = 15), and this response was blocked by the VIP receptor antagonist VIP6-28 (10–9 mol; P = 0.01, twoway ANOVA, n = 6–15). Vasoresponsiveness to VIP was absent in acutely inflamed joints (P = 0.2, one-way ANOVA, n = 5–7), but returned to normal in denervated inflamed joints (P = 0.0001, n = 8). MC stabilization with cromolyn did not alter synovial

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vasoresponsiveness to VIP in acutely inflamed knees (P = 0.74, n = 5–7). Conclusion. These findings suggest that the dilator effect of VIP in normal knee joints is attenuated during acute inflammation. This may be due to VIP receptor downregulation/desensitization caused by abundant endogenous VIP release from joint nerves, since the vasomotor effect of VIP returned following articular denervation. Finally, MCs do not contribute to VIPmediated vasoactivity in acutely inflamed knee joints. Characterization of GalR1, GalR2 and GalR3 immunoreactivity in catecholaminergic nuclei of the mouse brain Jessica J. Hawes, Marina R. Picciotto, Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA E-mail: [email protected] Aim of investigation. Distribution of the three identified galanin receptors, GalR1, GalR2 and GalR3, in areas of the brain involved in drug addiction is unknown. Our aim was to characterize immunoreactivity for the three identified galanin receptors, GalR1, GalR2 and GalR3, in parallel with galanin binding in catecholaminergic nuclei of the mouse brain, i.e., ventral tegmental area (VTA), nucleus accumbens (NA), substantia nigra (SN) and locus coeruleus (LC). Methods. Levels of GalR1, GalR2 and GalR3 were studied by western blotting, fluorescence immunohistochemistry, and infrared immunohistochemistry. Galanin binding sites were examined via [I125]-galanin equilibrium binding. Results. All three galanin receptors were found in the VTA, SN, NA, and LC. GalR1 protein is higher in the VTA, NA and SN than GalR2 and GalR3. GalR1 and GalR3 levels are high in the LC. The distribution of GalR1, GalR2 and GalR3 largely recapitulates the pattern of galanin binding throughout the brain, however some discrepancies exist. Conclusion. Our data suggest that GalR1 plays a role in regulation of dopamine neurotransmission within the VTA, NA and SN. GalR1 and GalR3 isoforms may be more critical than GalR2 for galanin-mediated regulation of noradrenergic. Overall, galanin receptors are distributed widely throughout the brain and all subtypes co-localize with tyrosine hydroxylase in dopaminergic and noradrenergic brain areas. Acknowledgement. This work was supported by the National Institutes of Health Grants DA15425. Reference Zachariou, V., Brunzell, D., Hawes, J., Stedman, D., Bartfai, T., Steiner, R., Wynick, D., Langel, U., Picciotto, M.R., 2003. The neuropeptide galanin modulates behavioral and neurochemical

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signs of opiate withdrawal. Proc. Natl. Acad. Sci. USA 100(15), 9028–33.

Effects of peptide histidine-isoleucine (PHI) and peptide histidine-methionine (PHM) on cAMP production in avian and mammalian cerebral cortex A. Dejda a, J.Z. Nowak a,b, a Centre of Medical Biology and Microbiology, Polish Academy of Sciences, Lodz, Poland, b Department of Pharmacology, Medical University, Lodz, Poland E-mail: [email protected] Aim of investigation. Our recent studies on biochemical effects of VIP and PACAP in the CNS of mammals and fowls have shown their potent stimulatory action on cAMP generation. As PHI, PHM and VIP are co-synthesized from the same precursor, share high level of amino acid similarity, and may act through common receptors, we asked whether PHI (rat and porcine) and PHM (human PHI) affect cAMP production in chick, rat and guinea pig cerebral cortex. Methods. Experiments were carried out on white leghorn chicks (Gallus domesticus), albino Wistar rats and guinea pigs (Dunkin Hurtley). The formation of [3H]cAMP and the effects of PHI and PHM were measured in [3H]adenine prelabelled slices of cerebral cortex. Results. All tested peptides (0.3–5 ?M) dose-dependently stimulated the synthesis of cerebral cAMP in rat and guinea pig, being significantly more potent in the latter species. In contrast to that, the peptides showed none or only weak activity in chicks. PACAP38 used as a reference drug at 0.1 ?M concentration was a strong stimulator of cAMP production in all experiments. Conclusion. Our results show that receptors mediating at least some biological effects of PHI and PHM in the CNS can be linked to AC/cAMP signaling pathway in mammals, but not in avians. Since PHI recognizes specific VIP/PACAP receptors in chick cerebral cortex, further study is needed to specify a signaling system through which this peptide realizes its action in the avian brain. Acknowledgement. The study was supported by the grants No 2 P05A 097 26 and No 3 P04C 076 25 from the State Committee for Scientific Research (KBN) in Poland. References Nowak, J.Z., Kuba, K., Zawilska ,J.B., 1999. Stimulatory effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic AMP formation in the hypothalamus and cerebral cortex of four avians and rat. Pol. J. Pharmacol. 51, 87–91. Nowak, J.Z., Kuba, K., 2001. Vasoactive intestinal peptide-stimulated adenosine 3’,5’-cyclic monophosphate formation in cerebral cortex and hypothalamus of chick and rat: comparison of the chicken and mammalian peptide. Neurosci. Lett. 297, 93–96.

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Activity-Dependent Neuroprotective Protein (ADNP) expression in mammalian cell lines and in rat brain N. Gennet a, V.J. Bubb b,c, A. Kipar d, J.P. Quinn a,b, a Department of Physiology, The University of Liverpool, UK, b Department of Human Anatomy and Cell Biology, The University of Liverpool, UK, c Department of Neurological Sciences, The University of Liverpool, UK, d Faculty of Veterinary Sciences The University of Liverpool, UK E-mail: [email protected] Aim. The function of ADNP is largely unknown. It has become a focus of interest because an 8 amino acid peptide, NAP, is able to convey astounding protective effects on neurons. Both in vivo and in vitro experiments confirm that NAP is able to protect neurons from damage in a variety of models. However, little is known about the role of full length ADNP. While the current model is that ADNP is proteolysed and secreted from glia, it also contains motifs indicating a role in gene regulation. The aim of this project is to investigate the function of ADNP by gene expression studies. Methods. Mammalian cell cultures are transfected with an ADNP expression construct. Morphology of the cells and cell numbers are monitored. Secondly, ADNP immunohistology is carried out in rat brains with chemically induced epilepsy. Results. ADNP over-expression in cells results in an increased cell number. ADNP transfected populations also have a ‘healthier’ phenotype. In the epilepsy model, necrotic cells seen in the CA1 region after induction of status epilepticus have lost ADNP immunoreactivity, while ‘healthy’ neurons show strong cytoplasmic and nuclear reaction. Conclusion. ADNP seems to affect cell number. The strength of the effect may not solely be due to an influence on the transfected cells, but may involve secretion of ADNP or proteolysed forms, thus affecting the whole population. Since an effect on cell number is not known for NAP, full length ADNP may have functions apart from those ascribed to NAP. Our in vitro data and the immunohistology indicate ADNP expression as a marker of a ‘healthy’ cell. Acknowledgements. This project is funded by the Wellcome Trust.

References Zamostiano, R. et al., 2001. Cloning and characterisation of human activity-dependent neuroprotective protein. J. Biol. Chem. 276(1), 708–14. Gozes, I. et al., 2003. From vasoactive intestinal peptide (VIP) through activity-dependent neuroprotective protein (ADNP) to NAP: a view of neuroprotection and cell division. J. Mol. Neurosci. 20(3), 315–22.

Distribution of peptidase activity in teleost and rat tissues J. Gil a, R. Laiz-Carrio´n b, J.M. Mancera b, J. Irazusta a, N. Agirregoitia a, a Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Bizkaia, Spain, b Department of Biology. Faculty of Sea and Environmental Sciences, University of. Ca´diz, Spain E-mail:[email protected] Aim of investigation. Peptides play important roles in cell regulation and signalling in many tissues. The actions of peptides are regulated by enzymes, known as peptidases, which are capable of degrading or processing them. Although the activity of these enzymes has been thoroughly characterized in mammals, little is know about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with similar tissues of the rat (lungs studied in place of gills). Methods. Enzyme activities were measured using bnaphthylamine amino acidic derivatives as substrates. Results. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase A (acid aminopeptidase) and B (basic aminopeptidase) were found in rat tissues, with the exception of aminopeptidase B in the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to trout. In contrast, the level of activity of aminopeptidase B was found to be consistently higher in all of the trout tissues analyzed in comparison to the sea bream (ST at least p < 0.05). Prolyl endoeptidase activity was principally dectected in the pituitary gland and in the brain areas of teleosts. Conclusion. Overall, these results indicate that peptidases have specific and important functions in teleosts. Acknowledgement. This work was supported by grants from the University of the Basque Country (00081.327-EA-7984/2000) and from the Jess GangoitiBarrera Foundation. N.A. was financially supported by a fellowship from the MEC (AP2000-3698).

Long-term hormonal effects of perinatal activation vs. blocking of endogenous cannabinoid receptors M. Moreno a, L. Escuredo b, R. Mun˜oz a, M. Navarro a, F. Rodriguez de Fonseca b, a Departamento. Psicobiologı´a, Facultad de Psicologı´a, Universidad Complutense de Madrid, Spain, b Fundacio´n Hospital Carlos Haya de Ma´laga, Spain E-mail: [email protected] Aim of investigation. Cannabis sativa preparations are the illicit drugs most commonly used by pregnant wo-

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men. Since cannabinoid CB1 receptors are present in HPA and HPG axis we suspected that the activation of the endogenous cannabinoid system during SNC developmental stages could induce long-term alterations in hormonal activity. Methods. The effects of perinatal exposure to the CB1 receptor agonist Delta-9-tetrahydrocannabinol (Delta9-THC) and the CB1 receptor antagonist SR141716A were studied measuring basal plasma levels of CRF, ACTH, corticosterone, LH and PRL hormone. Results. Perinatal exposure to Delta-9-THC produced an increment of CRF and corticosterone hormone in female rats, while male animals showed a decrement in the adult period. No significative changes where observed in LH and PRL hormone secretion. However, prenatal exposure to SR141716A induced significative changes in PRL hormone in the adulthood. These changes in hormonal secretion after perinatal activation or blocking of the endogenous cannabinoid system were accompanied also by behavioural alterations in the adult period of the animals. Conclusion. These long-term changes, showed with sexual dimorphism, indicate that the activation or blocking of the endogenous cannabinoid system during SNC plasticity stages, as perinatal period, affects HPA and HPG axis development inducing alterations in hormone secretion in the adulthood that could mediate on behavioural disturbances described after perinatal THC exposure. Acknowledgment. MCYT, FIS and Plan Nacional Sobre Drogas. Abbreviations. HPG, hipothalamus pituitary adrenal; HPG, hipothalamus pituitary gonadal; Delta-9-THC, Delta-9- Tetrahydrocannabinol; CRF, corticotropin releasing factor; ACTH, Adrenocorticotropic; LH, luteinizing hormone; PRL, prolactin hormone.

Aminopeptidase activity changes in the postmortem brain of human heroin addicts A. Varona a, J.J. Meana b, L.F. Callado b, A. Irazusta c, G. Larrinaga c, a Department of Physiology, University of the Basque Country, Leioa, Spain, b Department of Pharmacology, University of the Basque Country, Leioa, Spain, c Department of Nursing I, University of the Basque Country, Leioa, Spain E-mail: [email protected] Aim of investigation. Several studies have reported that the chronic administration of opioids induces changes in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the adult central nervous system. However, little is known about the catabolic regulation of opioid peptides and its contribution to neuroadaptative changes underlying drug addiction. In the present study, we have analyzed

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the activity of two enkephalin-degrading enzymes (puromycin-sensitive aminopeptidase or PSA and aminopeptidase N or APN) and two functionally different, soluble aminopeptidases (aminopeptidase B and aspartyl-aminopeptidase) in postmortem samples of prefrontal cortex and caudate nucleus of eight human heroin addict brains and eight matched-controls. Methods. Enzyme activities were fluorimetrically measured using -naphthylamide derivatives. Results. An increase in the activity of soluble PSA in the prefrontal cortex of heroin abusers was observed (heroin addict group: 51,452 + 3892 UAP/mg protein vs. control group: 42,003 + 2597 UAP/mg prot.; P < 0.05), while the activity of the other peptidases in both brain regions remained unaltered. Conclusion. This result agrees with previous findings in morphine-tolerant rats, and indicates that soluble PSA may be involved in neurobiological processes which underlie heroin addiction. Acknowledgement. This work was supported by grants from the University of the Basque Country (UPV/EHU 0081.327-EA-7984/2000) and MCYT (SAF 2002-10370 E). We thank the members of the Basque Institute of Legal Medicine for their cooperation.

Enkephalin-degrading enzymes and its relationship with the motility of the human sperm J. Irazusta a, A. Valdivia b, E. Agirregoitia a, C. Ochoa c, L. Casis a, a Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain, b Nursing Department II, Nursing School, University of the Basque Country, P Dr. J. Beguiristain, 105, 20018 San Sebastian, Gipuzkoa, Spain, c Laboratory of Seminology and Clinical Embryology, Euskalduna Clinic, c/ Euskalduna N 10-3, 48080 Bilbao, Bizkaia, Spain E-mail: [email protected] Aim of investigation. Opioid peptides have been reported to have important functions in human reproduction. Indeed, very high concentrations of enkephalins and their degrading enzymes have been descrived in human semen. In the present study, we compared the activity of two enkephalin degrading enzymes, aminopeptidase N (EC 3.4.11.2) and neutral endopeptidase 24.11 (EC 3.4.24.11), in different fractions of semen of semen from normozoospermic, fertile men and from subfertile patients with different abnormalities revealed by spermiogram analysis (asthenozoospermia, necrozoospermia and teratozoospermia). Methods. Enzyme activities were measured fluorimetrically. Aminopeptidase N activity using b-naphthylamine amino acidic derivatives as substrate and neutral

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endopeptidase activity using DAGNPG (dansyl-D-AlaGly-p-Nitro-Phe-Gly) as substrate. Results. High levels of activity of aminopeptidase N were found in the soluble (p < 0.001) and particulate (p < 0.05) sperm fractions of semen from patients presenting asthenozoospermia with necrozoospermia. In contrast, lower aminopeptidase N activity was measured in the soluble sperm fraction of asthenozoospermic semen. The percentage of immobile spermatozoa was negatively correlated with aminopeptidase activity in soluble (p < 0.001) and particulate (p < 0.05) sperm and prostasome (p < 0.05) fractions. Leves of activity of neutral endopeptidase were found to be unaltered among the different conditions. Conclusion. The results of the present study indicate that alterations in the activity of aminopeptidase N may be one of the molecular components which contribute to male human subfertility. Acknowledgement. This work was supported by grants from the University of the Basque Country (UPV/EHU, 00081.327-EA-4512/1998 and 1/UPV 00081.327-E-14891/2002) and from the Jess GangoitiBarrera Foundation. A. Valdivia and E. Agirregoitia were financially supported by a fellowship from the University of the Basque Country. Reaction of uterus-innervating sympathetic neurons in porcine inferior mesenteric ganglion to hysterectomyinduced axotomy K. Wasowicz Department of Functional Morphology, University of Warmia and Mazury, Olsztyn, Poland E-mail: [email protected]

In experimental animals either uterine horn or whole uterus were extirpated to evoke axotomy. In IMGs from experimental and control animals expression of TH, DBH, ChAT, NPY, GAL, VIP, SP as well as apoptosis(Bax, Bcl-2) and regeneration-associated (GAP-43) proteins was studied immunohistochemically, with in situ hybridization, as well as biochemically with RTPCR and immunoblotting. Result. Both in neurons innervating the uterine horn and uterine cervix a great drop in the number of THpositive neurons (from 90–95% to 34–42%) occurred. No change in the number of DBH and ChAT-positive ‘‘uterine neurons’’ was found. Hybridization in situ detected arrest of TH expression in axotomized neurons in IMG. No changes in the expression level of the enzymes were found with RT-PCR and immunoblotting. Axotomy evoked rise in the number of GAL-positive ‘‘uterine’’ neurons (from 0–8% to 71–73%), as well as of NPY-positive neurons (from 46–52% to 78–82%). The increase of expression at mRNA level was confirmed with in situ hybridization only in case of GAL. RT-PCR detected clear increase in the expression of GAL. No changes in the expression of Bax, Bcl-2 and GAP-43 were detected between control and experimental animals with immunohistochemistry, RT-PCR, or immunoblotting. Conclusion. No differences in the reaction to axotomy between neurons innervating the uterine horn and cervix could be found. The reaction was quite different from this described in laboratory animals.

Acknowledgements Aim of investigation. Sympathetic innervation of the uterus displays surprising degree of physiological plasticity during the estrus cycle and pregnancy. The behavior of this innervation is different in the uterine cervix and horn, where clear symptoms of nerve fibers degeneration are visible at the end of pregnancy. The aim of the study was to examine the response of uterine hornand cervix-innervating neurons of inferior mesenteric ganglion (IMG) to axotomy – the treatment usually evoking degeneration of nerve cells. Method. The study was done on sexually immature pigs. The neurons of IMG innervating the uterine horn and uterine cervix were identified with retrograde tracer.

Neuropeptides 2004 has been sponsored and supported by: Ministerio de Ciencia y Tecnologı´a (SAF200211881-E) Fundacio´n Bancaja Caja de Ahorros del Mediterra´neo (CAM) Universidad Miguel Herna´ndez de Elche Springer-Verlag, Berlı´n Novartis Menarini Ricerche IPSEN Patronato Municipal de Turismo de Alicante