Regulatory Peptides 192–193 (2014) 24–29

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Mediators involved in the hyperthermic action of neuromedin U in rats G. Telegdy a,b,⁎, A. Adamik b a b

Department of Pathophysiology, University of Szeged, Szeged, Hungary Neuroscience Research Group of the Hungarian Academy of Science, Faculty of Medicine, University of Szeged, Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 1 July 2013 Received in revised form 28 July 2014 Accepted 30 July 2014 Available online 7 August 2014 Keywords: Neuromedin U Hyperthermia Receptors

a b s t r a c t Neuromedin U (NmU), first was isolated from the porcine spinal cord, has subsequently been demonstrated in a number of species, in which it is present in the periphery and also the brain. Two receptors have been identified: NmU1R is mainly present in peripheral tissues, and Nmu2R in the central nervous system. NmU, a potent endogenous anorectic, serves as a catabolic signaling molecule in the brain; it inhibits food uptake, increases locomotion, activates stress mechanism, having cardiovasscular effects and, causes hyperthermia. The mechanism of this hyperthermia is unknown. In the present experiments, the effects of NmU on the colon temperature following i.c.v administration were studied in rats. For an investigation of the possible role of receptors in mediating hyperthermia, the animals were treated simultaneously with CRF 9–41 and antalarmin, a CRH1 receptor inhibitors, astressin 2B, a CRH2 receptor antagonist, haloperidol a dopamine receptor antagonist, atropine a muscarinic cholinergic receptor antagonist, noraminophenazone a cyclooxygenase inhibitor or isatin, a prostaglandin receptor antagonist. NmU increased the colon temperature, maximal action being observed at 2–3 h. CRF 9–41, antalarmin, astressin 2B haloperidol, atropine, noraminophenazone and isatin prevented the NmU-induced increase in colon temperature. The results demonstrated that, when injected into the lateral brain ventricle NmU increased the body temperature, mediated by CRHR1 and CRHR2, dopamine and muscarinic cholinergic receptors. The final pathway involves prostaglandin. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Neuromedin U (NmU), a member of the neuromedin family of peptides which includes bombesin-like, kassinin-like, neurotensin-like and neuromedin U (see for review, Malendowicz 2012)[1]. NmU which contract smooth muscle was originally isolated from the porcine spinal cord. Two forms have been identified with similar biological activities (NmU-25 and NmU-8) [2]. Mouse and rat NMU consist of 23 aminoacids [see Brighton et al. [3] for a review]. The evidence suggests roles for NmU in pain [4], in cancer [5], in the immune system [6], in the regulation of smooth muscle contraction in the gastrointestinal and genitourinary tracts [7] and in the control of blood flow and blood pressure [8–10]; NmU also decreases food intake and body weight, and increases gross locomotor activity, heat production, stress responses and body temperature [11]. It is a potent endogenous anorectic and serves as a catabolic signaling molecule in the brain [12]. More recently a new neuropeptide with 38 amino acids was

⁎ Corresponding author at: Department of Pathophysiology, Semmelweis 1., Szeged 6701, Hungary. Tel.: +36 62 545797; fax.: +36 62 545710. E-mail address: [email protected] (G. Telegdy).

http://dx.doi.org/10.1016/j.regpep.2014.07.004 0167-0115/© 2014 Elsevier B.V. All rights reserved.

isolated from rat brain and named as neuromedin S because it is mainly expressed in the suprachiasmatic nucleus of the hypothalamus [13] and is structurally related to NmU sharing a C-terminal core structure [14]. Two NmU receptors have been isolated: NmU-R1 and NmU-R2. The NmU-R1 is expressed in the periphery, and especially in the gastro-intestinal tract [15,16], whereas NmU-R2 is expressed predominantly in the CNS (hypothalamus, hippocampus, spinal cord, etc.) [15,17]. NmU increases the body temperature following central administration [12], but the possible involvement of neurotransmitters in mediating this action and the pathway leading to hyperthermia is unknown. In the present paper the action of the NmU-23 was studied on the colon temperature following administration in the brain ventricle in rats. The animals were treated either with NmU alone or in combination with different receptor blockers in doses which themselves could not influence the test [17,18]. The following receptor antagonists were tested: CRF 9–41 and antalarmin, a CRH1 receptor inhibitors, astressin 2B, a CRH2 receptor antagonist, haloperidol a dopamine receptor antagonist, atropine a muscarinic cholinergic receptor antagonist, noraminophenazone a cyclooxygenase inhibitor or isatin, a prostaglandin receptor antagonist.

G. Telegdy, A. Adamik / Regulatory Peptides 192–193 (2014) 24–29

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Fig. 1. The effect of different doses of NmU on colon temperature. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M. *p b 0.05, compared with the control group.

2. Material and methods 2.1. Animals Adult male Wistar rats weighing 200–240 g were used in the experiments. The animals were kept in groups of 5 or 6 in cages in a room at constant temperature (23 ± 0.5 °C) under a 12-h dark-light cycle (light on from 06.00 to 18.00 h) with free access to tap water and standard laboratory food. The animals were kept and handled during the experiments according to the protocol accepted by the Ethical Committee for the Protection of Animals in Research, University of Szeged, Hungary. All efforts were made to minimize animal suffering, to reduce the number of animals used.

Under pentobarbital (Nembutal Ceva, France; 35 mg/kg) intraperitoneal (i.p) anesthesia, the cannula was inserted stereotaxically into the lateral brain ventricle at a position with the coordinates 0.2 mm posterior and 1.7 mm lateral to the bregma, 3.7 mm deep from the dural surface, according to the atlas of Pellegrino et al. [19]. The cannula was fixed to the skull with dental cement and acrylate. The rats were allowed 5 days for recovery from surgery before the experimentation. Upon conclusion of the experiment, 10 μl methylene blue was injected into the ventricle of the decapitated animals and the brain was dissected to verify the appropriate positioning of the cannula. Only animals with the correct location of the cannula were used for evaluation of the experiment.

2.3. Materials 2.2. Surgery For the intracerebroventricular (i.c.v) administration of the NmU the rats were implanted with a 10-mm-long stainless steel cannula introduced into the lateral brain ventricle 5 days before the experiment.

Neuromedin U-23 and CRF (9–41) for i.c.v treatment were purchased from Bachem (Switzerland). Different doses of NMU were dissolved in sterile pyrogen-free 0.9% saline and injected in a volume of 2 μl.

Fig. 2. The effect of neuromedin U and CRF (9–41) on colon temperature. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

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Fig. 3. The effect of neuromedin U (1 μg/2 μl) and antalarmin (2 μg/2 μl) on the colon temperature. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M. * p b 0.05, compared with the control group.

The antalarmin and UCNR2 antagonist, astressin 2B were purchased from Sigma Aldrich Ltd., it was dissolved in saline and administered simultaneously with NmU 1 μg i.c.v. Antalarmin in a dose of 2 μg, astressin 2B 1 μg i.c.v, and atropine sulfate from EGYS (Budapest, Hungary) 1 mg kg i.m.; haloperidol from G. Richter (Budapest, Hungary) 40 μg kg i.m.; and Isatin was purchased from Sigma Chemical Co., St Louis, MO, USA., 25 mg/kg i.p. A pyrazolone derivative, noraminophenazone (Algopyrine; Chinoin, Hungary) was used to inhibit cyclooxygenase. Noraminophenazone was administered intramuscularly (i.m.) in a dose of 50 mg/kg. The effective doses of the antagonists were selected on the basis of previous experiences where the minimal doses were effective (in other tests), but did not themselves influence the tests [17,18].

subjected to handling by the experimenter each day after the cannulation. The room temperature was maintained at 23.0 ± 0.5 °C throughout the experiment. Each animal was then removed from the cage and gently restrained on the table with a cloth. The colon temperature was monitored by inserting the vaseline-lubricated thermistor probe of a digital electric thermometer (model: Cole-Parmer 8402-10) into the rectum of the animal. The experiments started at 8 a.m. with an initial colon temperature measurement. The following experiments were performed: The effects of centrally administered NMU 0.25, 0.5 and 1 μg with or without receptor antagonists on body temperature were measured. The most effective individual peptides were selected administered i.c.v in 1.0 μg. The colon temperature was measured before peptide administration, and 30 min 1, 2, 3, 4, 6 h after NmU or 1, 2, 3, 4, 6 h after NmU and receptor antagonist treatment.

2.4. Procedures On the day of the experiment, the animals were transferred to the laboratory 2 h before the beginning of the test in order for them to habituate to the experimental environment. In order to minimize the stress-induced increase in body temperature, the animals were

2.5. Statistical analysis Statistical analysis of the data was performed by analysis of variance (ANOVA). For significant ANOVA values, groups were compared by

Fig. 4. The effect of astressin 2B on neuromedin U-induced hyperthermia. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

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Fig. 5. The effect of haloperidol on neuromedin U-induced hyperthermia. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

Tukey's test for multiple comparisons with unequal cell size. A probability level of 0.05 was accepted as statistically relevant.

3. Results NmU in 0.25, 0.5 and 1 μg/2 μl i.c.v increased the body temperature steadily, and peaked at 2 h. At 6 h it was already declining,[F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,29) = 1.69, 3.17, 3.61, 27.51, 11.61, 16.90, 4.45] (Fig. 1). Simultaneous treatment with CRH (9–41)(1 μg/2 μl i.c.v.) fully blocked the hyperthermic action of NmU. [F30 min 1 h, 2 h, 3 h, 4 h, 6 h (3,18) = 0.59, 3.55, 16.41, 5.38, 5.79, 1.26] (Fig. 2). Simultaneous treatment with antalarmin (2 μg/2 μl i.c.v.) fully blocked or attenuated the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,39) = 1.26, 9.37, 31.07, 15.51, 13.11, 5.34] (Fig. 3). Simultaneous treatment with astressin 2B (1 μg/2 μl i.c.v.) fully blocked the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,18) = 0.20, 5.14, 32.08, 10.34, 4.55, 1.22] (Fig. 4).

Simultaneous treatment with haloperidol (40 μg/kg b.w.) fully blocked the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,18) = 0.98, 8.84, 23.97, 20.25, 10.26, 0.43] (Fig. 5). Simultaneous treatment with atropine (1 mg/kg b.w.) fully blocked the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,19) = 4.43, 7.56, 29.44, 2.99, 2.66, 1.67] (Fig. 6). Simultaneous treatment with noraminophenazone (50 mg/kg b.w.) fully blocked the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,20) = 4.18, 4.37, 24, 37, 24.14, 38.39, 12.02, 6.05] (Fig. 7). Simultaneous treatment with isatin (25 mg/kg b.w.) fully blocked the hyperthermic action of NmU. [F30 min, 1 h, 2 h, 3 h, 4 h, 6 h (3,18) = 0.39, 25.28, 26.16, 4.02, 2.34, 0.88] (Fig. 8). 4. Discussion We earlier demonstrated that a number of neuropeptides such as ANP, BNP, CNP [20] and PACAP [21], can increase body temperature. Various other neuropeptides are also involved in thermo-regulation, e.g. cholecystokinin [22], ACTH, arginine vasopressin, melanocyte-

Fig. 6. The effect of atropine on neuromedin U-induced hyperthermia. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

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Fig. 7. The effect of noraminophenazone on neuromedin U-induced hyperthermia. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

stimulating hormone [23], TRH [24], galanin [25,26], and neuropeptide Y [27]. Urocortin 1,(UCN 1), UCN 2 and UCN 3 administered into the lateral brain ventricle all cause hyperthermia [28]. The specific receptor for UCN 1 is the CRF 1 receptor which is the receptor for CRF too. For UCN 2 and UCN 3, the specific receptor is the CRF 2 receptor. In the present work we have confirmed the hyperthermic action of NmU following i.c.v administration [12]. It has also been reported that NmU activates the pituitary–adrenal system, in which the action of CRF [29] and vasopressin could be involved [30]. Both CRF and vasopressin increase the body temperature [23] and CRF is responsible for cytokine-induced hyperthermia [31] and stress-induced hyperthermia [32], We have now demonstrated that CRF1 receptor antagonists (CRF 9–41 and antalarmine) can block the hyperthermic action of NmU. It is interesting to note that CRF2 receptor is also involved in the hyperthermic action of NmU since the CRF 2 receptor antagonist astressin 2B similarly blocked the hyperthermic action of NmU. It is not clear whether NmU is acting directly on the CRF 2 receptor or stimulating either UCN 2 or UCN3 or both in this way activating the CRF2 receptor. The action of

NmU was also blocked with antagonists of classical transmitter antagonist haloperidol, a D2 antagonist. We demonstrated earlier that the hyperthermic action of PACAP was likewise blocked by haloperidol [21]. In our experiments, NmU-induced hyperthermia was also blocked by atropine. It has an antagonist of the muscarinic and nicotinic cholinergic mechanism [33]. The hyperthermic action of a number of neuropeptides was prevented by a cyclooxygenase inhibitor blocking prostaglandin synthesis [21,34]. The hyperthermic action of NmU can additionally be blocked by treatment with the cyclooxygenase inhibitor noraminophenazone, demonstrating the involvement of a cascade mechanism in which a participant in the common final pathway is prostaglandin E2 [35]. The involvement of prostaglandin in this hyperthermic action is strengthened by the finding that isatin, which inhibited PGE2-induced fever in other experiments [36,37] also blocks NmU-induced hyperthermia. Conflict of interest The authors declare that there are no conflicts of interest.

Fig. 8. The effect of isatin on neuromedin U-induced hyperthermia. Number of animals per group is presented in parenthesis after the corresponding group. The vertical lines on the top of the marks denote the S.E.M., * p b 0.05, compared with the control group.

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Acknowledgments Supported by: TAMOP 4.2.1/B-09 and the Hungarian Academy of Sciences. References [1] Malendowicz LK, Ziolkowska A, Rucinski M. Meuromedins U and S involvement in the regulation of the hypothalamo-pituitary–adrenal axis. Front Endocrinol 2012; 3:1–10. [2] Minamino N, Kangawa K, Matsuo H. Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptide identified in porcine spinal cord. Biochem Biophys Res Commun 1985;130:1078–85. [3] Brighton PJ, Szekeres PG, Willars GB. Neuromedin U and its receptors:structure, function and physiological roles. Pharmacol Rev 2004;56:231–48. [4] Nakahara K, Kojima M, Hanada R, Egi Y, Ida T, et al. Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice Biochem. Biophys Res Commun 2004;15:615–20. [5] Alevizios I, Mahadevappa M, Zhang X, Ohyama H, Kohno Y, et al. Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis. Oncogene 2001;20:6196–204. [6] Hedrick JA, Morse K, Shan LX, Qiao XD, Pang L, et al. Identification of a human gastrointestinal tract and immune system receptor for neuromedin U. Mol Pharmacol 2000;58:870–5. [7] Westfall TD, McCafferty GP, Pullen M, Gruver S, Sulpizio AC, et al. Characterization of neuromedin U effects in canine smooth-muscle. J Pharmacol Exp Ther 2001;301: 987–92. [8] Minamino N, Kangawa K, Fukuda M, Matsuo H. Neuromedin-L — a novel mammalian tachykinin identified in porcine spinal-cord. Neuropeptides 1984;4:157–66. [9] Chu CP, Jin QH, Kunitake T, Kato K, Nabekura T, Nakazato M, et al. Cardiovascular actions of central neuromedin U in conscious rats. Regul Pept 2002;105:29–34. [10] Rahman AA, Shahid IZ, Pilowsky PM. Neuromedin U causes biphasic cardiovascular effects and impairs baroreflex function in rostral ventrolateral medulla of spontaneously hypertensive rat. Peptides 2013;44:15–24. [11] Hanada R, Nakazato M, Murakami K, Sakihara S, Yoshimatsu H, et al. The role for neuromedin U in stress response. Biochem Biophys Res Commun 2001;289:225–8. [12] Nakazato M, Hanada R, Murakami N, Date Y, Mondal MS, et al. Central effects of neuromedin U in regulation of energy homeostasis. Biochem Biophys Res Commun 2000;277:191–4. [13] Mori K, Miyazato M, Ida T, Murakami N, Serino R, Ueta Y, Kojima M, Kangawa K. Identification of neuromedin s and its possible role in the mammalianncircadian oscillatr system. EMBO J 2005;24:325–35. [14] Mori K, Miyazato M, Kangawa K. Neuromedin S: discovery and functions. Results Probl Cell Differ 2008;46:201–12. [15] Domin J, Ghatei MA, Chohan P, Bloom SR. Neuromedin U — a study of its distribution in the rat. Peptides 1987;8:779–84. [16] Steel JH, Vannoorden S, Ballesta J, Gibson SJ, Ghatei MA, et al. Localization of 7B2, neuromedin-B and neuromedin-U in specific cell-types of rat, mouse and human pituitary in rat hypothalamus and in 30 human pituitary and extrapituitary tumors. Endocrinology 1988;122:270–82.

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[17] Mangold C, Ksiazek I, Yun CW, Berger E, Binkert C. Distribution of neuromedin U binding sites in the rat CNS revealed by in vitro receptor autoradiography. Neuropeptides 2008: 377–86. [18] Telegdy G, Tiricz H, Adamik A. Involvement of neurotransmitters in urocortininduced passive avoidance learning in mice. Brain Res Bull 2005;67:242–7. [19] Tanaka M, Csabafi K, Telegdy G. Neurotransmissions of antidepressant-like effects of kisspeptin-13. Regul Pept 2012;228:388–91. [20] Pellegrino IJ, Pellegrino AS, Cushman AJ. Stereotaxic atlas of the rat brain. New York: Plenum Press; 1979 8–57. [21] Pataki I, Jászberényi M, Telegdy G. Hyperthermic action of centrally administered natriuretic peptide in the rat. Pept 1999;20:193–7. [22] Pataki I, Adamik A, Jászberényi M, Mácsai M, Telegdy G. Pituitary adenylate cyclaseactivating polypeptide induces hyperthermia in the rat. Neuropharmacology 2000; 39:1303–8. [23] Székely M. The vagus nerve in thermoregulation and energy metabolism. Auton Neurosci 2000;85:26–38. [24] Ehymayed HM, Jansky L. A discrete mode of antipyretic action of AVP, alpha-MSH and ACTH. Physiol Res 1992;41:57–61. [25] Arancibia S, Rage F, Astier H, Tapia-Arancibia L. Neuroendocrine and autonomous mechanism underlying thermoregulation in cold environment. Neuroendocrinology 1996;64:257–67. [26] Maurelli M, Marchini E, Tartara A. EEG and autonomic effects of centrally administered galanin in the rabbit. Boll Soc Ital Biol Sper 1993;9:485–91. [27] Patel S, Hutson PH. Hypothermia induced by cholinomimetic drugs is blocked by galanin possible involvement of ATP-sensitive K + channels. Eur J Pharmacol 1994;255:26–32. [28] Billington CJ, Levine AS. Hypothalamic neuropeptide Y regulation of feeding and energy metabolism. Curr Opin Neurobiol 1992;2:847–51. [29] Telegdy G, Adamik A, Toth G. The action of urocortins on body temperature in rats. Peptides 2006;27:2289–94. [30] Hanada T, Date Y, Shimbara T, Sakihara S, Murakami N, et al. Central actions of neuromedin U via corticotrophin-releasing hormone Biochem. Biophys Res Commun 2003;954:958. [31] Wren AM, Small CJ, Abbott CR, Jethwa PH, Kennedy AR, et al. Hypothalamic actions of neuromedin U. Endocrinology 2002;143:4227–34. [32] Strijbos PJ, Hardwick AJ, Relton JK, Carey F, Rothwell NJ. Inhibition of central actions of cytokines on fever and thermogenesis by lipocortin-1 involves CRF. Am J Physiol 1992;263:E632–6. [33] Nakamori T, Morimoto A, Murakami N. Effects of a central CRF antagonist on cardiovascular and thermoregulatory responses induced by stress or IL.1 beta. Am J Physiol 1993;265:R834–9. [34] Simpson CW, Ruwe WD, Myers RD. Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation. Neuroscience. Biobehav Rev 1994;18(1):1–20. [35] Pataki I, Adamik A, Jászberényi M, Mácsai M, Telegdy G. Involvement of transmitters in pituitary adenylate cyclase-activating polypeptide-induced hyperthermia. Regul Pept 2003;15:187–93. [36] Rothwell NJ. Central activation of thermogenesis by prostaglandins: dependence on CRF. Horm Metab Res 1990;22:616–8. [37] Telegdy G, Adamik A, Glover V. Antipyretic action of isatin and its analogues in mice and rats. Neurosci Med 2011;2:1–5.

Mediators involved in the hyperthermic action of neuromedin U in rats.

Neuromedin U (NmU), first was isolated from the porcine spinal cord, has subsequently been demonstrated in a number of species, in which it is present...
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