Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

peptides 29 (2008) 1183–1190

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Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice Yi-qing Wang a, Jia Guo a, Sheng-bin Wang a, Quan Fang a, Feng He a, Rui Wang a,b,* a

Key Laboratory of Preclinical Study for New Drugs of Gansu Province, and Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China b State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China

article info

abstract

Article history:

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide

Received 23 January 2008

FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important

Received in revised form

roles in thermoregulation, which suggested a link between opioid receptors and NPFF

25 February 2008

receptors in the production of hypothermia. Therefore, we designed a study to investigate

Accepted 26 February 2008

the relationship between opioid and NPFF in control of thermoregulation in mice. The

Published on line 4 March 2008

selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol)

Keywords:

after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third

Opioid

ventricle reduced the hypothermia induced by morphine (5 nmol,) or nociceptin/orphanin

Nociceptin/orphanin FQ (N/OFQ)

FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol)

Morphine

nor NOP receptor antagonist [Nphe1]NC(1-13)NH2 (7.5 nmol) reduced the hypothermia

Neuropeptide FF (NPFF)

induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose

RF9

of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not

Hypothermia

modified. These results suggest that NPFF receptors activation is required for opioid to

Mice

produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals. # 2008 Elsevier Inc. All rights reserved.

1.

Introduction

Opioid receptors are currently classified as classical (MOP, DOP and KOP receptors) and nonclassical (NOP receptor) [40]. Opioid receptors were involved in the physiological control of numerous functions of the central nervous system, including thermoregulation. It has long been recognized that opioid such as morphine could produce a range of effects on body

temperature in a number of species including man [1,37,41]. The effect on body temperature of morphine, which acted on the classical opioid receptors, were biphasic in mice, with low doses producing hyperthermia and higher doses resulting in hypothermia [3,12,32,44]. The hypothermia induced by morphine could be antagonized by the opioid receptor antagonists naloxone (classical opioid receptors antagonist), naloxonazine (MOP receptor antagonist), BNTX (DOP receptor antagonist)

* Corresponding author at: Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China. Tel.: +86 931 8912567/852 34003755; fax: +86 931 8911255/852 23649932. E-mail addresses: [email protected], [email protected] (R. Wang). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.02.016

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and DIPPA (KOP receptor antagonist), which provided further evidence for the involvement of classical opioid receptors [3,33]. N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, enhanced the hypothermic effects of morphine in mice [38]. In addition, NOP receptor is the fourth member of the opioid receptor family [20]. Nociceptin/ orphanin FQ (N/OFQ) isolated from the mammal brain, a 17amino-acid peptide, was an endogenous ligand of the NOP receptor [19,29]. N/OFQ decreased body temperature in rats, which was reduced by NOP receptor antisense oligonucleotides treatment in rats [5,14,28,39,42]. Neuropeptide FF (NPFF, FLFQPQRF-NH2) belongs to a family of amidated neuropeptides related to the molluscan cardioexcitatory neuropeptide FMRFamide (FMRFa) [25]. NPFF immunoreactivity was localized in different CNS sites involved in temperature regulation [2,7]. In thermoregulatory study, injection into the third ventricle of NPFF or 1DMe (a stable NPFF analog) elicited a marked decrease in basal rectal temperature [6,8,10,11,27]. L-NAME markedly potentiated hypothermia induced by 1DMe injected in the mouse brain [45]. The previous studies indicated that NPFF might act as a modulator of endogenous opioid functions [22,23,24,34,35,43]. At the cellular level, NPFF receptors induced anti-opioid actions [34]. In vivo, the studies of NPFF were mostly focused on the pro- and anti-opioid effects on morphine antinociception or morphine tolerance and dependence [16,17,18,26]. However, the possible interactions between NPFF and opioid agonists in the mediation of hypothermic effects are not very clear and need more investigations. Both opioid and NPFF systems were involved in thermoregulation of the rodents. NO participated, in the same manner, in hypothermia evoked by NPFF and morphine [38,45]. Therefore, these data suggested a link between opioid and NPFF systems in body temperature regulation. In the present study, we used the endpoint of hypothermia to investigate opioid and NPFF systems interactions in conscious mice.

2.

Materials and methods

2.1.

Animals

Male Kunming strain mice were obtained from the Experimental Animal Center of Lanzhou University. All animals were cared for and experiments were carried out in accordance with the European Community guidelines for the use of experimental animals (86/609/EEC). All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China.

2.2.

Chemicals

NPFF, RF9, N/OFQ and [Nphe1]NC(1-13)NH2 were synthesized on a solid-phase support following the previous report [9]. Peptides were prepared by manual solid-phase synthesis using standard Fmoc chemistry. N-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids (ACT and Fluka, USA) were coupled to a Rink Amide MBHA resin (Tianjin Nankai

Hecheng Science & Technology Co., Ltd., China). The following schedule was employed: (1) DMF wash (3); (2) 20% piperidine/DMF (3, 4 min); (3) DMF wash (3); (4) NaFmoc-Amino Acid (2.5 equiv.)/HBTU (2.5 equiv.)/HOBt (2.5 equiv.)/DIPEA (5 equiv.) in DMF (1), 1 h; (5) DMF wash (3); (6) Kaiser Test. The N-acylated residue of RF9 was added to the resin bound peptide using 1-adamantanecarboxylic acid (3 equiv.)/HBTU (3 equiv.)/HOBt (3 equiv.)/DIPEA (6 equiv.) in DMF (1), 1 h. The protected peptide-resin was treated with reagent K (TFA/H2O/phenol/ethanedithiol/ thioanisole, 82.5:5:5:2.5:5) for 2 h at room temperature. Gel filtration was performed to desalt the crude peptides. The purity of the peptide was checked by reversed-phase highperformance liquid chromatography (HPLC). Analytical HPLC analyses were performed on a Waters Delta 600 system coupled to a UV detector with a Delta-pak Analytical ˚ , 5 mm). column C18 (3.9 mm  150 mm, 300 A Morphine hydrochloride was the product of Shenyang First Pharmaceutical Factory, China. Naloxone hydrochloride was obtained from Tocris Cookson, Bristol, UK. All drugs were dissolved in saline and stored at 20 8C.

2.3.

Temperature measurement

Male Kunming mice weighing 27–30 g were used. The mice were placed in the specially designed restraining device as described by Rosow et al., with their tails taped lightly to horizontal posts [32]. Each animal was used only once. The ambient temperature was regulated to 21  0.5 8C and relative humidity of 52  2%. The experiments were performed between 10:00 and 14:00 h. Rectal temperature was measured with a thermistor probe (Machine Equipment Corporation of GaoBeiDian, China) inserted to a depth of 2.5 cm into the rectum, which was linked to a recorder system (model BL-420E+, Taimeng Technology Corporation of Chengdu, China). The i.c.v. administration in the third ventricle was performed following the method described by France´s et al. [11]. The drugs were co-injected to investigate whether the hypothermic effects of the agonists could be antagonized by RF9 or other. Body temperature was recorded before injection and then at 15, 30, 45, 60, 90 and 120 min after i.c.v. in the third ventricle of control or various treatments. Changes in body temperature after injection and before drug administration were calculated for each animal. The time courses of change in body temperature of mice subjected to different treatments are shown in the figure. The raw data from each animal were converted to area under the curve (AUC). We calculated the AUC data for the interval of time in which agonist induced clear changes in body temperature, namely 0–60 min. Data obtained under the various treatments were statistically compared by means of one-way ANOVA followed by the Bonferroni’s post hoc test.

2.4.

Statistical analysis

Data were given as means  S.E.M. One-way ANOVA followed by the Bonferroni’s post hoc test was used to establish statistical significance; a probability level of P < 0.05 was considered to be significant.

peptides 29 (2008) 1183–1190

Fig. 1 – (A) Time course of the change of body temperature induced by morphine in the absence or presence of RF9 injected into the third ventricle in mice. (B) Time course of the change of body temperature induced by NPFF in the absence or presence of naloxone injected into the third ventricle in mice. The rectal temperature was recorded after injection of vehicle control, agonists, or the coapplication of antagonists and agonists. Data points represent means W S.E.M. from experiments conducted on 8–16 mice per group. The statistical analysis was described in the text and Fig. 2.

3.

Results

3.1.

Morphine–NPFF interaction

As shown in Figs. 1A and 2A, central injection of morphine (5 nmol) evoked the decrease in body temperature (AUC: 108.2  7.8; P < 0.001, vs. saline), which was blocked by coinjection of naloxone (10 nmol) (P < 0.01, vs. 5 nmol morphine group) (Fig. 2A). At a dose of 45 nmol, NPFF produced significant hypothermia following injection into the third ventricle (AUC: 87.9  16.1; P < 0.01, vs. saline) (Figs. 1B and 2B). The hypothermic effects of NPFF (45 nmol) were fully prevented by co-injection of RF9 (30 nmol) (P < 0.01, vs. 45 nmol of NPFF group) (Fig. 2B). The effects of RF9 (30 nmol)

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Fig. 2 – (A) Inhibitory effects of RF9 (20, 30 nmol), naloxone (10 nmol) co-injected i.c.v. in the third ventricle on the hypothermia of morphine (5 nmol) in mouse. Data are expressed as differences in area under the curve between morphine (5 nmol) alone and morphine co-injected with RF9, naloxone, WS.E.M. during 0–60 min. P < 0.01 indicating significant differences from the hypothermia of morphine (5 nmol) in the absence of RF9 or nalone (oneway ANOVA followed by the Bonferroni’s post hoc test). (B) Inhibitory effects of RF9 (30 nmol), naloxone (10 nmol) coinjected i.c.v. in the third ventricle on the hypothermia of NPFF (45 nmol) in mouse. Data are expressed as differences in area under the curve between NPFF (45 nmol) and NPFF co-injected with RF9, naloxone, WS.E.M. during 0–60 min. P < 0.01 indicating significant difference from the hypothermia of NPFF (45 nmol) in the absence or presence of RF9; P > 0.05 indicating no statistically significant difference from the hypothermia of NPFF (45 nmol) in the absence or presence of nalone (oneway ANOVA followed by the Bonferroni’s post hoc test).

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Table 1 – Effect of morphine N/OFQ and NPFF on body temperature Treatment

Area under the curve

Morphine 5 nmol 2 nmol 0.5 nmol

108.2  7.8 47.5  11.8 17.6  9.7

N/OFQ 2 nmol 1 nmol 0.5 nmol

119.3  11.2 47.6  10.1 21.3  6.3

NPFF 45 nmol 30 nmol 5 nmol

87.9  16.1 46.3  6.5 21.5  8.2

Co-injection 5 nmol morphine + 0.5 nmol N/OFQ 5 nmol morphine + 5 nmol NPFF 2 nmol N/OFQ + 0.5 nmol morphine 2 nmol N/OFQ + 5 nmol NPFF 45 nmol NPFF + 0.5 nmol morphine 45 nmol NPFF + 0.5 nmol N/OFQ

28.7  6.7** 94.8  18.8 138.9  27.5 125.7  12 81.7  19.3 77.3  5.9

The area under the curve (S.E.M.) was calculated for the 1-h period following the third ventricle injection. **P < 0.01 vs. morphine alone at the same dose (one-way ANOVA followed by the Bonferroni’s post hoc test).

co-injected with an effective dose of morphine (5 nmol) did not affect the morphine-induced hypothermic effects.

3.2.

Fig. 3 – (A) Effect of RF9 (20, 30 nmol) co-injected i.c.v. in the third ventricle on the hypothermia of N/OFQ (2 nmol) in mouse. (B) Effect of [Nphe1]NC(1-13)NH2 (7.5 nmol) coinjected i.c.v. in the third ventricle on the hypothermia of NPFF (45 nmol) in mouse. Data points represent means W S.E.M. from experiments conducted on 8–16 mice. Area under the curve (AUC) during 0–60 min calculated from these data are statistically analyzed and are presented in the text and Fig. 4.

and naloxone (10 nmol) injected into the third ventricle on body temperature did not differ significantly from the effect of saline (Fig. 1A and B). As shown in Fig. 2A, it was worthy to note that RF9 (30 nmol) significantly reduced the hypothermia induced by morphine (5 nmol) (AUC: 53.9  11.6; P < 0.01 vs. 108.2  7.8 for 5 nmol morphine). RF9 (20 nmol) slightly, but not significantly, reduced morphine-induced hypothermia (P > 0.05) (Fig. 2A). However, naloxone (10 nmol) did not modify the hypothermia of NPFF (45 nmol) (P > 0.05, vs. 45 nmol NPFF) (Fig. 2B). As shown in Table 1, co-injected with a low dose of morphine (0.5 nmol), the hypothermia of NPFF (45 nmol) was not modified. Similarly, a low dose of NPFF (5 nmol)

N/OFQ–NPFF interaction

The decrease in body temperature induced by N/OFQ (2 nmol) was maximal at 15 min after injection into the third ventricle (AUC: 119.2  11.2; P < 0.001 vs. saline group) (Fig. 3A). As shown in Fig. 4A, [Nphe1]NC(1-13)NH2 (7.5 nmol) prevented the hypothermic response to N/OFQ (2 nmol) (P < 0.01). Compared to saline (n = 8), central injection of [Nphe1]NC(1-13)NH2 (7.5 nmol) did not change body temperature (Fig. 3B). The hypothermic effects of N/OFQ (2 nmol) was significantly reduced by the third ventricle administration of RF9 (30 nmol) (AUC: 59.7  10.7; P < 0.01 vs. 119.2  11.2 for 2 nmol N/OFQ) (Fig. 4A). RF9 (20 nmol) slightly, but not significantly, decreased N/OFQ-induced hypothermia (P > 0.05) (Fig. 4A). However, [Nphe1]NC(1-13)NH2 (7.5 nmol) did not change the hypothermia induced by NPFF (45 nmol) (P > 0.05, vs. 45 nmol NPFF) (Fig. 4B). As shown in Table 1, compared to the 45 nmol NPFF, a low dose of N/OFQ (0.5 nmol) did not influence the hypothermia induced by an effective dose of NPFF (45 nmol). Similarly, a low dose of NPFF (5 nmol) had no effect on the hypothermia induced by an effective dose of N/OFQ (2 nmol).

3.3.

Morphine–N/OFQ interaction

As shown in Fig. 5, naloxone (10 nmol) had no effect on the N/ OFQ-induced hypothermia (P > 0.05). In addition, [Nphe1]NC(113)NH2 (7.5 nmol) did not modify morphine-induced hypothermia (P > 0.05) (Fig. 5). Interestingly, as shown in Table 1, a low dose of N/OFQ (0.5 nmol) significantly inhibited hypothermia

peptides 29 (2008) 1183–1190

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Fig. 5 – Effects of [Nphe1]NC(1-13)NH2 (7.5 nmol) co-injected i.c.v. in the third ventricle on the hypothermic responses evoked by morphine (5 nmol) and effects of naloxone (10 nmol) co-injected i.c.v. in the third ventricle on the hypothermic responses evoked by N/OFQ (2 nmol). Data were presented as means W S.E.M. from experiments conducted on 8–16 mice.

4.

Fig. 4 – (A) Inhibitory effects of RF9 (20, 30 nmol), [Nphe1]NC(1-13)NH2 (7.5 nmol) co-injected i.c.v. in the third ventricle on the hypothermia of N/OFQ (2 nmol) in mouse. Data are expressed as differences in area under the curve between N/OFQ (2 nmol) alone and N/OFQ coinjected with RF9, [Nphe1]NC(1-13)NH2, WS.E.M. during 0– 60 min. P < 0.01 indicating significant differences from the hypothermia of N/OFQ (2 nmol) in the absence of RF9 or [Nphe1]NC(1-13)NH2 (one-way ANOVA followed by the Bonferroni’s post hoc test). (B) Inhibitory effects of RF9 (30 nmol), [Nphe1]NC(1-13)NH2 (7.5 nmol) co-injected i.c.v. in the third ventricle on the hypothermia of NPFF (45 nmol) in mouse. Data are expressed as differences in area under the curve between NPFF (45 nmol) alone and NPFF coinjected with RF9, [Nphe1]NC(1-13)NH2, WS.E.M. during 0– 60 min. P < 0.01 indicating significant differences from hypothermia of NPFF (45 nmol) in the absence or presence of RF9; P > 0.05 indicating no statistically significant difference from the hypothermia of NPFF (45 nmol) in the absence or presence of [Nphe1]NC(1-13)NH2 (one-way ANOVA followed by the Bonferroni’s post hoc test).

induced by morphine (5 nmol) (AUC: 28.7  6.7; P < 0.01 vs. 108.2  7.8 for 5 nmol morphine), while a low dose of morphine (0.5 nmol) did not modify the hypothermic effects of N/OFQ (2 nmol) (P > 0.05).

Discussion

The major finding of the present study was that the NPFF receptors antagonist RF9 attenuated the hypothermia caused by two different opioid agonists, morphine and N/OFQ. In contrast, the hypothermia evoked by the central administration of NPFF was not affected by opioid antagonists. The previous studies indicated that NPFF might act as a modulator of opioid functions [22–24,34,35,43]. In rodents, NPFF receptors agonists exhibited either anti-opioid activities or potentiate opioid analgesia when injected intracerebroventricularly or intrathecally, respectively [35]. Prior work demonstrated that NPFF receptors induced anti-opioid actions at the cellular level [34]. The studies concerning the NPFF were mostly focused on their pro- and anti-opioid effects on morphine antinociception or morphine tolerance and dependence [16,17,18,26]. However, the possible interactions between NPFF and opioid agonists in the mediation of the hypothermic effects were poorly understood. In this study, we selected agonists and antagonists of opioid and NPFF systems to investigate the possible interactions between two systems. In this study, NPFF injected into the third ventricle elicited a marked decrease in basal rectal temperature, in agreement with the previous findings [6,8,10,11,27]. NPFF receptors antagonist RF9 showed antagonistic effects on NPFF receptors in vitro and in vivo studies [15,36]. The present data were consistent with our previous study, 30 nmol RF9, which had no effect alone, completely antagonized the hypothermic effects of NPFF [10]. Previous studies from our laboratory and those of our colleagues indicated that BIBP3226, a mixed antagonist of NPY Y1 and NPFF receptors, could fully prevent NPFF-induced hypothermia [8]. The hypothermia evoked by the central administration of NPFF was not affected by naloxone (classical

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opioid receptors antagonist) similarly to that previously observed [6]. In our study, [Nphe1]NC(1-13)NH2 had no effect on NPFF-evoked hypothermia. A low dose of N/OFQ (0.5 nmol) co-injected with an effective dose of NPFF (45 nmol) did not change the hypothermia of the NPFF. Similarly, a low dose of morphine (0.5 nmol) co-injected with an effective dose of NPFF (45 nmol) did not modify the hypothermic effects of the NPFF, which kept agreement with the prior report [6]. These results suggest that NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. Consistent with previous studies, morphine, which acted on the classical opioid receptors, caused a significant hypothermia in mice [3,12,30,32,44]. The hypothermia induced by morphine could be antagonized by the opioid receptor antagonists naloxone (classical opioid receptors antagonist), naloxonazine (MOP receptor antagonist), BNTX (DOP receptor antagonist) and DIPPA (KOP receptor antagonist), which provided further evidence for the involvement of classical opioid receptors [3,33]. Interestingly, 30 nmol RF9 reduced a significant proportion of the hypothermia induced by morphine. In addition, a low dose of NPFF (5 nmol) co-injected with an effective dose of morphine (5 nmol) did not affect the morphine-induced hypothermic effects similarly to that previously observed [6]. These results suggest that NPFF receptors activation is required for morphine to produce hypothermia in the mouse brain. NOP receptor, the fourth member of the opioid receptor family, was similar to MOP, DOP and KOP receptors (65% homology) [19,20,29]. In our experiments, the central administration of N/OFQ produced a significant hypothermia, which was in accord with the recent deduction [5,14,28,39,42]. Prior work has established that Ro64-6198, a NOP receptor agonist, caused hypothermia in wild type mice but not in NOP receptor knockout mice [14]. The direct injection of N/OFQ into the hypothalamus produced a rapid hypothermia, similar to our findings following N/OFQ administration into the third ventricle [42]. A possible central site of action was the hypothalamus, the major thermoregulatory center in the brain [4]. [Nphe1]NC(1-13)NH2, a pure and selective NOP receptor antagonist, did not affect body temperature [13]. Our results extended those studies by showing that antagonism of NOP receptors by [Nphe1]NC(1-13)NH2 decreased the hypothermic response to N/OFQ. The effect of [Nphe1]NC(113)NH2 provided pharmacological evidence that N/OFQ activated NOP receptor to produce hypothermia. Similarly, 30 nmol RF9 attenuated N/OFQ-induced hypothermia in the mouse brain. A low dose of NPFF (5 nmol) co-injected with an effective dose of N/OFQ (2 nmol) did not affect the N/OFQinduced hypothermia. These results suggest that NPFF receptors activation is necessary for N/OFQ to produce hypothermia in the mouse brain. In a word, NPFF and its analogs exhibited either anti-opioid or pro-opioid effects on morphine antinociception, tolerance and dependence [16,17,18,26]. To our surprise, the NPFF receptors antagonist RF9 but not NPFF reduced a significant proportion of the hypothermia induced by opioid. It was worthy to note that the dose of RF9 that attenuated opioidevoked hypothermia was equivalent to the dose which blocked the hypothermic response to NPFF. These data

suggest that pharmacological antagonism of NPFF receptors decrease a significant percentage of the hypothermia caused by opioid agonists. Furthermore, these findings suggest that opioid and NPFF receptors interactions in the CNS are functionally significant. One possible mechanism may be that changes in NPFF receptors signaling mediate part of the hypothermia caused by opioid agonists. The observation that NO participates, in the same manner, in hypothermia evoked by NPFF and morphine support it [38,45]. The site of the drug interaction between opioid and RF9 is unknown, but the hypothalamus is a possibility. Still another possibility that must be considered is a pharmacokinetic interaction between RF9 and opioid agonists. The physiological mechanism responsible for the interaction between opioid agonist and RF9 is unknown. Potential physiological causes of drug-induced hypothermia are a decrease in heat production, increase in heat loss, or a lowered setpoint of thermoregulation [31]. Future studies will determine the physiological interaction between opioid and NPFF systems by investigating additional physiological parameters, such as motor activity, pain transmission, oxygen consumption and skin temperature. Furthermore, in our study, naloxone had no effect on the hypothermia induced by N/OFQ in mice. [Nphe1]NC(1-13)NH2 did not modify morphine-induced hypothermia in mice. These suggest that the N/OFQ receptor fails to bind opioid ligands and N/OFQ fails to bind to opioid receptors [21]. Prior work demonstrated that OFQ/N blocked morphine-induced hyperthermia in rats [5]. In our study, a low dose of N/OFQ could decrease a significant proportion of the hypothermia caused by an effective dose of morphine in mice. These results suggest that the mechanism of N/OFQ- and morphine-induced hypothermia are different, N/OFQ probably acts as a physiological antagonist to reduce morphine-induced change of body temperature in rodents. Additionally, it was worthy to note that RF9 shortened the duration of N/OFQ-induced hypothermia whereas it decreased the amplitude of hypothermia induced by morphine in the present study. These observations agree with the above-mentioned deduction that morphine and N/OFQ exerted hypothermic action via different pathways. However, it is difficult to explain the phenomenon at present, further research of mechanism are expected. In conclusion, the balance between receptor systems in the brain contributes to body temperature regulation. Two of the most important of these systems are the opioid and NPFF receptors systems. We demonstrated that opioid-evoked hypothermia was attenuated by a NPFF receptors antagonist. It is concluded that indirect NPFF receptors mechanism(s) may be involved in the opioid-induced hypothermia in mice. The exact mechanism is not yet apparent, but cross-talk between opioid and NPFF receptors may exert a significant influence on body temperature.

Acknowledgements This study was supported by the grants from the National Natural Science Foundation of China (Nos. 20525206, 20772052 and 20621091), the Specialized Research Fund for the Doctoral Program in Higher Education Institutions (No. 20060730017),

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and the Chang Jiang Program of the Ministry of Education of China.

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