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Orexin 2 receptor is involved in orexin A-induced hyperlocomotion in rats
Pharmacological Reports 71 (2019) 1147–1150
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Orexin 2 receptor is involved in orexin A-induced hyperlocomotion in rats Shunsuke Maehara* , Junji Furukawa, Takumi Ota Pharmacology, Research Center, Mochida Pharmaceutical Co., Ltd., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2019 Received in revised form 10 June 2019 Accepted 29 June 2019 Available online 16 July 2019
Background: The orexin system regulates various functions, including sleep/wake cycles, feeding, and cognition. Orexin A and orexin B are endogenous neuropeptides for both orexin 1 (OX1) and orexin 2 (OX2) receptors. Orexin A has a potent agonistic activity for both the receptors and is known to increase locomotor activity in rats. However, it has not been elucidated how each receptor contributes to orexin A-induced hyperlocomotion. Methods: We examined the effects of an OX1 receptor antagonist, SB 334867, and an OX2 receptor antagonist, EMPA, as well as an OX1 and OX2 receptor antagonist on hyperlocomotion caused by intracerebroventricular administration of orexin A or an OX2 receptor agonist, ADL-OXB ([Ala11,D-Leu15]-orexin B), in rats. Results: EMPA (100 mg/kg, ip) but not SB 334867 (3–10 mg/kg, ip) showed antagonistic effects on ADL-OXBinduced hyperlocomotion without affecting the spontaneous locomotor activity. Both EMPA (100 mg/kg, ip) and the OX1 and OX2 receptor antagonist (3–30 mg/kg, po) antagonized orexin A-induced hyperlocomotion, while SB 334867 (3–10 mg/kg, ip) showed no effects. Conclusions: Our results suggest that orexin A-induced hyperlocomotion is mainly mediated by the activation of the OX2 receptor. © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
Keywords: Orexin A OX1 receptor OX2 receptor Locomotor activity
Introduction Orexin A and orexin B, also known as hypocretin 1 and hypocretin 2, are endogenous neuropeptides, which have been identified as ligands of orphan G protein-coupled receptors obtained from rat brain extracts [1]. Accumulating evidence indicates that orexins are involved in the regulation of multiple functions including feeding [1], sleep/wake cycles [2,3], and addiction [4,5]. The physiological functions of orexins are exerted via two receptor subtypes, orexin 1 (OX1) receptor and orexin 2 (OX2) receptor, which belong to a family of G protein-coupled receptor. Orexin A is a 33-residue peptide and exhibits potent agonistic activity against both OX1 and OX2 receptors [1]. On the contrary, orexin B consists of 28-amino acids and has a 10-fold higher affinity for the OX2 receptor than for the OX1 receptor [1]. The expression patterns of the two receptors in the brain partially overlap, and some brain regions express only one type of receptor
* Corresponding author. E-mail address: [email protected] (S. Maehara).
[6]. Therefore, each receptor is likely to have different pharmacological functions [7]. Orexin A is known to cause increased locomotor activity after intracerebroventricular administration in rats [8–10]. Because it has been reported that orexin A promotes wakefulness [8] and stimulates food consumption [1] in rats, the hyperlocomotion in response to orexin A may reflect the increased arousal and seeking behavior. However, it has not yet been sufficiently clarified how each receptor contributes to orexin A-induced hyperlocomotion. Samson et al. reported that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124, partially blocked orexin A-induced hyperlocomotion, but the OX2 receptor agonist, ADLOXB ([Ala11,D-Leu15]-orexin B), did not show these effects in rats [10]. However, the effect of an OX2 receptor antagonist has not been confirmed in orexin A-induced hyperlocomotion yet. In the present study, we examined the effects of an OX1 receptor antagonist, SB 334867, and an OX2 receptor antagonist, EMPA on an OX1 and OX2 receptor agonist, orexin A-induced hyperlocomotion in rats and compared with those of an OX1 and OX2 receptor antagonist. In addition, we examined the effects of SB 334867 and EMPA on the OX2 receptor agonist, ADL-OXBinduced hyperlocomotion in rats.
http://dx.doi.org/10.1016/j.pharep.2019.06.018 1734-1140/© 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
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S. Maehara et al. / Pharmacological Reports 71 (2019) 1147–1150
Materials and methods
injection cannula was left in place for 30 s before removal to avoid liquid backflow.
Animals ADL-OXB- or orexin A-induced hyperlocomotion Male Sprague-Dawley rats (6 weeks old, Charles River Laboratories Japan, Inc., Kanagawa, Japan) were purchased. All rats were group-housed before surgery and were individually housed after surgery in an air-conditioned room (room temperature; 23 3 C, humidity; 55 15%), with a 12-h light-dark cycle (lights on: 05:3017:30). Each rat was provided with standard chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and tap water ad libitum. The rats were allowed to acclimatize in the facility for at least 7 days prior to initiation of the experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee complied with the Japanese law of “Act on Welfare and Management of Animals” and the guidelines of the Ministry of Health, Labor and Welfare of Japan.
The locomotor activity was measured using a digital counter system with an infrared sensor (SUPERMEX, Muromachi Kikai Co, Ltd., Tokyo, Japan). Each rat was placed into a novel plastic cage (width, 270 mm depth, 440 mm height, 187 mm) with clean paper chips and habituated to the plastic cage for 120 min before administration of the test compounds. SB 334867, EMPA, or OX1 and OX2 receptor antagonist were administered to each rat at 30, 30, or 60 min before administration of ADL-OXB (3 nmol/rat, icv) or orexin A (1 nmol/rat, icv). The spontaneous locomotor activity was measured in the plastic cage. Immediately after administration of ADL-OXB or orexin A, each rat was returned to the plastic cage and the locomotor activity was measured for 120 min.
Drugs Statistical analysis OX1 receptor antagonist, SB 334867 (N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea, Tocris Bioscience, Bristol, UK) and an OX 2 receptor antagonist, EMPA (N-Ethyl-2[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]N-(3-pyridinylmethyl)-acetamide, Chinglu Pharmaceutical Research LLC, Newington, CT, USA) were suspended in 0.5% methylcellulose solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and intraperitoneally (ip) administered at a volume of 5 mL/kg. An OX1 and OX2 receptor antagonist, suvorexant (Haoyuan Chemexpress Co., Ltd., Shanghai, China) was suspended in 20% TPGS (D-α-tocopherol polyethylene glycol 1000 succinate, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) in distilled water with gentle warming and orally (po) administered at a volume of 5 mL/kg. An OX2 receptor agonist, ADL-OXB ([Ala11, 15 D-Leu ]-orexin B, Tocris Bioscience, Bristol, UK), orexin A (Tocris Bioscience, Bristol, UK), and angiotensin II (Peptide Institute, Inc., Osaka, Japan) were dissolved in physiological saline and intracerebroventricularly (icv) administered at a volume of 5 mL/rat. Based on our preliminary studies, we selected the doses of 1 and 3 nmol/rat for ADL-OXB and orexin A, respectively.
Student’s t-test was used to analyze the effects of ADL-OXB and orexin A for the locomotor activity. The effects of the test compounds were analyzed using one-way analysis of variance and post-hoccomparisons were performed using Dunnett’s test. A probability level of < 0.05 was considered statistically significant. Results Effects of SB 334867 and EMPA on ADL-OXB-induced hyperlocomotion in rats We examined the effects of SB 334867 and EMPA on ADL-OXBinduced hyperlocomotion in rats. EMPA significantly antagonized ADL-OXB-induced hyperlocomotion at a dose of 100 mg/kg, ip, while SB 334867 had no effects at doses of 3 and 10 mg/kg, ip (Fig. 1). Neither compounds affected the spontaneous locomotor activity at all tested doses (Table 1).
Surgery The rats were placed on a stereotaxic frame (NARISHIGE Group, Tokyo, Japan) under anesthesia with pentobarbital sodium (50 mg/ kg, ip). For icv administration of ADL-OXB or orexin A, a guide cannula (21 G, 10 mm) was implanted at 0.9 mm posterior, 1.2 mm lateral, 2.3 mm ventral to the bregma and was secured with screws and dental cement, and all rats were individually housed after surgery. The total number of rats undergone surgery was 154 in the present study. After a recovery period of at least 7 days after surgery, the placement of the guide cannula was tested by injection of angiotensin II (100 ng/rat, icv). Because angiotensin II is known to induce drinking behavior within a few minutes [11], water consumption was measured for 30 min. Only rats displaying consumption of at least 5 g of water were selected and used for the subsequent experiments. Intracerebroventricular administration The injection cannula (25 G, 13 mm) was connected to a microsyringe (Hamilton Company, NV, USA) through polyethylene tubing and inserted into the guide cannula. The compound or vehicle was infused to each rat via the injection cannula by gently pushing the free-moving syringe piston. After infusion, the
Fig. 1. Effects of SB 334867 and EMPA on ADL-OXB-induced hyperlocomotion in rats. SB 334867 (3–10 mg/kg, ip) or EMPA (30–100 mg/kg, ip) was administered 30 min before the administration of ADL-OXB (3 nmol/rat, icv). ADL-OXB-induced hyperlocomotion was measured as the total activity during a 120 min observation period immediately after intracerebroventricular administration of ADL-OXB. Data are expressed as the mean SEM of 6 animals. Sharps represent a significant difference from the vehicle + vehicle-treated group, ##p < 0.01 (Student’s t-test). Asterisks represent a significant difference from the vehicle + ADL-OXB-treated group, **p < 0.01 (Dunnett’s test).
S. Maehara et al. / Pharmacological Reports 71 (2019) 1147–1150 Table 1 Effects of SB 334867 and EMPA on spontaneous locomotor activity in rats. Drug (mg/kg, ip) + drug (nmol/rat, icv)
Locomotor activity (activity counts/30 min)
Vehicle + vehicle Vehicle + ADL-OXB (3) SB 334867 (3) + ADL-OXB (3) SB 334867 (10) + ADL-OXB (3) EMPA (30) + ADL-OXB (3) EMPA (100) + ADL-OXB (3)
1732 172 2383 544 2699 569 2462 429 1895 224 1397 346
Data are expressed as the mean SEM of 6 animals.
Effects of SB 334867, EMPA, and OX1 and OX2 receptor antagonist on orexin A-induced hyperlocomotion in rats The effects of SB 334867, EMPA, and OX1 and OX2 receptor antagonist against orexin A-induced hyperlocomotion were tested in rats. EMPA showed significant antagonistic effects against orexin A-induced hyperlocomotion at a dose of 100 mg/kg, ip, while SB 334867 had no effects at doses of 3 and 10 mg/kg, ip (Fig. 2A). The OX1 and OX2 receptor antagonist significantly antagonized orexin A-induced hyperlocomotion at all tested doses (Fig. 2B). Discussion In the present study, we found that the OX2 receptor antagonist, EMPA, antagonized an OX1 and OX2 receptor agonist, orexin Ainduced hyperlocomotion in rats as well as the OX1 and OX2 receptor antagonist. On the other hand, the OX1 receptor antagonist, SB 334867, showed no effects. In addition, we showed that EMPA but not SB 334867 antagonized the hyperlocomotion caused by an OX2 receptor agonist, ADL-OXB in rats and that these compounds did not affect the spontaneous locomotor activity at all tested doses. These results indicate that orexin A-induced hyperlocomotion may be mediated mainly by the activation of the OX2 receptor. ADL-OXB is reported as a selective OX2 receptor agonist with 400fold selectivity for OX2 receptor (EC50 0.13 nM) over OX1 receptor (EC50 52 nM) [12]. It can be also used as a pharmacological tool to investigate the role of the OX2 receptor. Because ADL-OXB has been shown to cause hyperlocomotion in rats [10], we first examined the effective dose of the OX2 receptor antagonist, EMPA to antagonize ADL-induced hyperlocomotion in rats. EMPA significantly showed antagonistic effects on ADL-induced hyperlocomotion at a dose of
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100 mg. Of note, EMPA did not affect the spontaneous locomotor activity at a dose of 100 mg/kg. This suggests that the observed EMPA-mediated antagonism of ADL-OXB-induced hyperlocomotion is a specific pharmacological effect by the blockade of OX2 receptor in vivo. On the other hand, an OX1 receptor antagonist, SB 334867 did not antagonize ADL-OXB-induced hyperlocomotion. This result was nearly consistent with the previous report that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124 did not antagonize ADL-OXB-induced hyperlocomotion [10]. Orexin A is a dual agonist for OX1 and OX2 receptors [1] and has been shown to cause hyperlocomotion in freely moving rats [8–10]. Therefore, we examined the effects of EMPA and SB 334867 as well as an OX1 and OX2 receptor antagonist on orexin A-induced hyperlocomotion in rats. EMPA significantly antagonized orexin A-induced hyperlocomotion at a dose of 100 mg/kg, which was equivalent to the effective dose against ADL-OXB-induced hyperlocomotion. These results indicate that the activation of the OX2 receptor is involved in orexin A-induced hyperlocomotion. On the other hand, SB 334867 had no effects on orexin A-induced hyperlocomotion at all tested doses. Our present results were a somewhat inconsistent with a previous report which showed that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124 partially blocked the total behavioral activity caused by orexin A in rats [10]. This discrepancy might be due to the differences in the compound itself and the route of administration of OX1 receptor antagonist between the two studies. We administered SB 334867 intraperitoneally, while Samson et al. administered SB 408124 intracerebroventricularly [10]. Because SB 408124 poorly crosses the blood-brain barrier (brain-to-plasma ratio 0.02) compared to SB 334867 (brain-toplasma ratio 0.29) [13], SB 334867 is intensively used for systemic administration in in vivo experiments in contrast to the OX1 receptor antagonist, SB 408124, although both the compounds had comparable antagonistic activity for the OX1 receptor [13,14]. In the present study, we selected the doses of SB 334867 as 3 and 10 mg/kg. Previously, it was reported that SB 334867 achieved about 80–90% occupancy for OX1 receptor from 30 min to 2 h at a subcutaneous dose of 10 mg/kg using ex vivo occupancy study in rats [13]. Taken together this finding with the profile that SB 334867 is an OX1 receptor antagonist with only approximately 50-fold selectivity over OX2 receptor [13], SB 334867 may also block OX2 receptor in addition to OX1 receptor at higher doses than 10 mg/kg. However, because we did not examine occupancy for OX2 receptor at various doses of SB 334867 in the present study, we could not
Fig. 2. Effects of SB 334867 (A), EMPA (A), and OX1 and OX2 receptor antagonist (B) on orexin A-induced hyperlocomotion in rats. SB 334867 (3–10 mg/kg, ip) or EMPA (30– 100 mg/kg, ip) was administered 30 min before administration of orexin A (1 nmol/rat, icv). The OX1 and OX2 receptor antagonist (3–30 mg/kg, po) was administered 1 h before the administration of orexin A. Orexin A-induced hyperlocomotion was measured as the total activity during a 120 min observation period immediately after administration of orexin A. Data are expressed as the mean SEM of 6 animals. Sharps represent a significant difference from the vehicle + vehicle-treated group, ##p < 0.01 (Student’s t-test). Asterisks represent a significant difference from the vehicle + orexin A-treated group, *p < 0.05, **p < 0.01 (Dunnett’s test).
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assess whether higher doses of SB 334867 affect orexin A-induced hyperlocomoion by the blockade of OX2 receptor. Our results indicate that the activation of the OX1 receptor may not make a major contribution to orexin A-induced hyperlocomotion. However, further studies with other systemically available OX1 receptor antagonists with high selectivity over OX2 receptor will be necessary to clarify the role of OX1 receptor in orexin A-induced hyperlocomotion. In addition, the effects of an OX1 and OX2 receptor antagonist in orexin A-induced hyperlocomotion were tested. The OX1 and OX2 receptor antagonist as well as EMPA antagonized orexin A-induced hyperlocomotion in a dose-dependent manner. These results suggest that the blockade of the OX2 receptor may be mainly involved in the antagonistic effect of the OX1 and OX2 receptor antagonist against orexin A-induced hyperlocomotion. In summary, we demonstrated that an OX2 receptor antagonist but not an OX1 receptor antagonist attenuated the hyperlocomotion caused by orexin A in rats. These results suggest that orexin Ainduced hyperlocomotion is mainly mediated by the activation of the OX2 receptor.
[2]
[3]
[4]
[5]
[6] [7]
[8]
[9]
[10]
Conflict of interest [11]
None. Acknowledgments
[12]
We thank Natsuko Yuge and Tatsuya Hoshino for their assistance in the in vivo experiments.
[13]
References
[14]
[1] Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G
protein-coupled receptors that regulate feeding behavior. Cell 1998;92 (4):573–85. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, et al. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 1999;98(3):365–76. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, et al. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 1999;98(4):437–51. Ghaemi-Jandabi M, Azizi H, Ahmadi-Soleimani SM, Semnanian S. Intracoerulear microinjection of orexin-A induces morphine withdrawallike signs in rats. Brain Res Bull 2017;130:107–11. James MH, Stopper CM, Zimmer BA, Koll NE, Bowrey HE, Aton-Jones G. Increased number and activity of a lateral subpopulation of hypothalamic orexin/hypocretin neurons underlies the expression of an addicted state in rats. Biol Psychiatry 2019;85:925–35. Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 1998;438(12):71–5. Tsujino N, Sakurai T. Orexin/hypocretin: a neuropeptide at the interface of sleep, energy homeostasis, and reward system. Pharmacol Rev 2009;61 (2):162–76. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, et al. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci 1999;96(19):10911–6. Nakamura T, Uramura K, Nambu T, Yada T, Goto K, Yanagisawa M, et al. Orexininduced hyperlocomotion and stereotypy are mediated by the dopaminergic system. Brain Res 2000;873(1):181–7. Samson WK, Bagley SL, Ferguson AV, White MM. Orexin receptor subtype activation and locomotor behavior in the rat. Acta Physiol (Oxf) 2010;198 (3):313–24. Simpson JB, Mangiapane ML, Dellmann HD. Central receptor sites for angiotensin-induced drinking: a critical review. Fed Proc 1978;37 (13):2676–82. Asahi S, Egashira S, Matsuda M, Iwaasa H, Kanatani A, Ohkubo M, et al. Development of an orexin-2 receptor selective agonist, [Ala11,D-Leu15]. Bioorg Med Chem Lett 2003;13(1):111–3. Bonaventure P, Yun S, Johnson PL, Shekhar A, Fitz SD, Shireman BT, et al. A selective orexin-1 receptor antagonist attenuates stress-induced hyperarousal without hypnotic effects. J Pharmacol Exp Ther 2015;352 (3):590–601. Langmead CJ, Jerman JC, Brough SJ, Scott C, Porter RA, Herdon HJ. Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor. Br J Pharmacol 2004;141 (2):340–6.
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Short communication
Orexin 2 receptor is involved in orexin A-induced hyperlocomotion in rats Shunsuke Maehara* , Junji Furukawa, Takumi Ota Pharmacology, Research Center, Mochida Pharmaceutical Co., Ltd., 722 Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2019 Received in revised form 10 June 2019 Accepted 29 June 2019 Available online 16 July 2019
Background: The orexin system regulates various functions, including sleep/wake cycles, feeding, and cognition. Orexin A and orexin B are endogenous neuropeptides for both orexin 1 (OX1) and orexin 2 (OX2) receptors. Orexin A has a potent agonistic activity for both the receptors and is known to increase locomotor activity in rats. However, it has not been elucidated how each receptor contributes to orexin A-induced hyperlocomotion. Methods: We examined the effects of an OX1 receptor antagonist, SB 334867, and an OX2 receptor antagonist, EMPA, as well as an OX1 and OX2 receptor antagonist on hyperlocomotion caused by intracerebroventricular administration of orexin A or an OX2 receptor agonist, ADL-OXB ([Ala11,D-Leu15]-orexin B), in rats. Results: EMPA (100 mg/kg, ip) but not SB 334867 (3–10 mg/kg, ip) showed antagonistic effects on ADL-OXBinduced hyperlocomotion without affecting the spontaneous locomotor activity. Both EMPA (100 mg/kg, ip) and the OX1 and OX2 receptor antagonist (3–30 mg/kg, po) antagonized orexin A-induced hyperlocomotion, while SB 334867 (3–10 mg/kg, ip) showed no effects. Conclusions: Our results suggest that orexin A-induced hyperlocomotion is mainly mediated by the activation of the OX2 receptor. © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
Keywords: Orexin A OX1 receptor OX2 receptor Locomotor activity
Introduction Orexin A and orexin B, also known as hypocretin 1 and hypocretin 2, are endogenous neuropeptides, which have been identified as ligands of orphan G protein-coupled receptors obtained from rat brain extracts [1]. Accumulating evidence indicates that orexins are involved in the regulation of multiple functions including feeding [1], sleep/wake cycles [2,3], and addiction [4,5]. The physiological functions of orexins are exerted via two receptor subtypes, orexin 1 (OX1) receptor and orexin 2 (OX2) receptor, which belong to a family of G protein-coupled receptor. Orexin A is a 33-residue peptide and exhibits potent agonistic activity against both OX1 and OX2 receptors [1]. On the contrary, orexin B consists of 28-amino acids and has a 10-fold higher affinity for the OX2 receptor than for the OX1 receptor [1]. The expression patterns of the two receptors in the brain partially overlap, and some brain regions express only one type of receptor
* Corresponding author. E-mail address: [email protected] (S. Maehara).
[6]. Therefore, each receptor is likely to have different pharmacological functions [7]. Orexin A is known to cause increased locomotor activity after intracerebroventricular administration in rats [8–10]. Because it has been reported that orexin A promotes wakefulness [8] and stimulates food consumption [1] in rats, the hyperlocomotion in response to orexin A may reflect the increased arousal and seeking behavior. However, it has not yet been sufficiently clarified how each receptor contributes to orexin A-induced hyperlocomotion. Samson et al. reported that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124, partially blocked orexin A-induced hyperlocomotion, but the OX2 receptor agonist, ADLOXB ([Ala11,D-Leu15]-orexin B), did not show these effects in rats [10]. However, the effect of an OX2 receptor antagonist has not been confirmed in orexin A-induced hyperlocomotion yet. In the present study, we examined the effects of an OX1 receptor antagonist, SB 334867, and an OX2 receptor antagonist, EMPA on an OX1 and OX2 receptor agonist, orexin A-induced hyperlocomotion in rats and compared with those of an OX1 and OX2 receptor antagonist. In addition, we examined the effects of SB 334867 and EMPA on the OX2 receptor agonist, ADL-OXBinduced hyperlocomotion in rats.
http://dx.doi.org/10.1016/j.pharep.2019.06.018 1734-1140/© 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.
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Materials and methods
injection cannula was left in place for 30 s before removal to avoid liquid backflow.
Animals ADL-OXB- or orexin A-induced hyperlocomotion Male Sprague-Dawley rats (6 weeks old, Charles River Laboratories Japan, Inc., Kanagawa, Japan) were purchased. All rats were group-housed before surgery and were individually housed after surgery in an air-conditioned room (room temperature; 23 3 C, humidity; 55 15%), with a 12-h light-dark cycle (lights on: 05:3017:30). Each rat was provided with standard chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) and tap water ad libitum. The rats were allowed to acclimatize in the facility for at least 7 days prior to initiation of the experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee complied with the Japanese law of “Act on Welfare and Management of Animals” and the guidelines of the Ministry of Health, Labor and Welfare of Japan.
The locomotor activity was measured using a digital counter system with an infrared sensor (SUPERMEX, Muromachi Kikai Co, Ltd., Tokyo, Japan). Each rat was placed into a novel plastic cage (width, 270 mm depth, 440 mm height, 187 mm) with clean paper chips and habituated to the plastic cage for 120 min before administration of the test compounds. SB 334867, EMPA, or OX1 and OX2 receptor antagonist were administered to each rat at 30, 30, or 60 min before administration of ADL-OXB (3 nmol/rat, icv) or orexin A (1 nmol/rat, icv). The spontaneous locomotor activity was measured in the plastic cage. Immediately after administration of ADL-OXB or orexin A, each rat was returned to the plastic cage and the locomotor activity was measured for 120 min.
Drugs Statistical analysis OX1 receptor antagonist, SB 334867 (N-(2-Methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea, Tocris Bioscience, Bristol, UK) and an OX 2 receptor antagonist, EMPA (N-Ethyl-2[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]N-(3-pyridinylmethyl)-acetamide, Chinglu Pharmaceutical Research LLC, Newington, CT, USA) were suspended in 0.5% methylcellulose solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and intraperitoneally (ip) administered at a volume of 5 mL/kg. An OX1 and OX2 receptor antagonist, suvorexant (Haoyuan Chemexpress Co., Ltd., Shanghai, China) was suspended in 20% TPGS (D-α-tocopherol polyethylene glycol 1000 succinate, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) in distilled water with gentle warming and orally (po) administered at a volume of 5 mL/kg. An OX2 receptor agonist, ADL-OXB ([Ala11, 15 D-Leu ]-orexin B, Tocris Bioscience, Bristol, UK), orexin A (Tocris Bioscience, Bristol, UK), and angiotensin II (Peptide Institute, Inc., Osaka, Japan) were dissolved in physiological saline and intracerebroventricularly (icv) administered at a volume of 5 mL/rat. Based on our preliminary studies, we selected the doses of 1 and 3 nmol/rat for ADL-OXB and orexin A, respectively.
Student’s t-test was used to analyze the effects of ADL-OXB and orexin A for the locomotor activity. The effects of the test compounds were analyzed using one-way analysis of variance and post-hoccomparisons were performed using Dunnett’s test. A probability level of < 0.05 was considered statistically significant. Results Effects of SB 334867 and EMPA on ADL-OXB-induced hyperlocomotion in rats We examined the effects of SB 334867 and EMPA on ADL-OXBinduced hyperlocomotion in rats. EMPA significantly antagonized ADL-OXB-induced hyperlocomotion at a dose of 100 mg/kg, ip, while SB 334867 had no effects at doses of 3 and 10 mg/kg, ip (Fig. 1). Neither compounds affected the spontaneous locomotor activity at all tested doses (Table 1).
Surgery The rats were placed on a stereotaxic frame (NARISHIGE Group, Tokyo, Japan) under anesthesia with pentobarbital sodium (50 mg/ kg, ip). For icv administration of ADL-OXB or orexin A, a guide cannula (21 G, 10 mm) was implanted at 0.9 mm posterior, 1.2 mm lateral, 2.3 mm ventral to the bregma and was secured with screws and dental cement, and all rats were individually housed after surgery. The total number of rats undergone surgery was 154 in the present study. After a recovery period of at least 7 days after surgery, the placement of the guide cannula was tested by injection of angiotensin II (100 ng/rat, icv). Because angiotensin II is known to induce drinking behavior within a few minutes [11], water consumption was measured for 30 min. Only rats displaying consumption of at least 5 g of water were selected and used for the subsequent experiments. Intracerebroventricular administration The injection cannula (25 G, 13 mm) was connected to a microsyringe (Hamilton Company, NV, USA) through polyethylene tubing and inserted into the guide cannula. The compound or vehicle was infused to each rat via the injection cannula by gently pushing the free-moving syringe piston. After infusion, the
Fig. 1. Effects of SB 334867 and EMPA on ADL-OXB-induced hyperlocomotion in rats. SB 334867 (3–10 mg/kg, ip) or EMPA (30–100 mg/kg, ip) was administered 30 min before the administration of ADL-OXB (3 nmol/rat, icv). ADL-OXB-induced hyperlocomotion was measured as the total activity during a 120 min observation period immediately after intracerebroventricular administration of ADL-OXB. Data are expressed as the mean SEM of 6 animals. Sharps represent a significant difference from the vehicle + vehicle-treated group, ##p < 0.01 (Student’s t-test). Asterisks represent a significant difference from the vehicle + ADL-OXB-treated group, **p < 0.01 (Dunnett’s test).
S. Maehara et al. / Pharmacological Reports 71 (2019) 1147–1150 Table 1 Effects of SB 334867 and EMPA on spontaneous locomotor activity in rats. Drug (mg/kg, ip) + drug (nmol/rat, icv)
Locomotor activity (activity counts/30 min)
Vehicle + vehicle Vehicle + ADL-OXB (3) SB 334867 (3) + ADL-OXB (3) SB 334867 (10) + ADL-OXB (3) EMPA (30) + ADL-OXB (3) EMPA (100) + ADL-OXB (3)
1732 172 2383 544 2699 569 2462 429 1895 224 1397 346
Data are expressed as the mean SEM of 6 animals.
Effects of SB 334867, EMPA, and OX1 and OX2 receptor antagonist on orexin A-induced hyperlocomotion in rats The effects of SB 334867, EMPA, and OX1 and OX2 receptor antagonist against orexin A-induced hyperlocomotion were tested in rats. EMPA showed significant antagonistic effects against orexin A-induced hyperlocomotion at a dose of 100 mg/kg, ip, while SB 334867 had no effects at doses of 3 and 10 mg/kg, ip (Fig. 2A). The OX1 and OX2 receptor antagonist significantly antagonized orexin A-induced hyperlocomotion at all tested doses (Fig. 2B). Discussion In the present study, we found that the OX2 receptor antagonist, EMPA, antagonized an OX1 and OX2 receptor agonist, orexin Ainduced hyperlocomotion in rats as well as the OX1 and OX2 receptor antagonist. On the other hand, the OX1 receptor antagonist, SB 334867, showed no effects. In addition, we showed that EMPA but not SB 334867 antagonized the hyperlocomotion caused by an OX2 receptor agonist, ADL-OXB in rats and that these compounds did not affect the spontaneous locomotor activity at all tested doses. These results indicate that orexin A-induced hyperlocomotion may be mediated mainly by the activation of the OX2 receptor. ADL-OXB is reported as a selective OX2 receptor agonist with 400fold selectivity for OX2 receptor (EC50 0.13 nM) over OX1 receptor (EC50 52 nM) [12]. It can be also used as a pharmacological tool to investigate the role of the OX2 receptor. Because ADL-OXB has been shown to cause hyperlocomotion in rats [10], we first examined the effective dose of the OX2 receptor antagonist, EMPA to antagonize ADL-induced hyperlocomotion in rats. EMPA significantly showed antagonistic effects on ADL-induced hyperlocomotion at a dose of
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100 mg. Of note, EMPA did not affect the spontaneous locomotor activity at a dose of 100 mg/kg. This suggests that the observed EMPA-mediated antagonism of ADL-OXB-induced hyperlocomotion is a specific pharmacological effect by the blockade of OX2 receptor in vivo. On the other hand, an OX1 receptor antagonist, SB 334867 did not antagonize ADL-OXB-induced hyperlocomotion. This result was nearly consistent with the previous report that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124 did not antagonize ADL-OXB-induced hyperlocomotion [10]. Orexin A is a dual agonist for OX1 and OX2 receptors [1] and has been shown to cause hyperlocomotion in freely moving rats [8–10]. Therefore, we examined the effects of EMPA and SB 334867 as well as an OX1 and OX2 receptor antagonist on orexin A-induced hyperlocomotion in rats. EMPA significantly antagonized orexin A-induced hyperlocomotion at a dose of 100 mg/kg, which was equivalent to the effective dose against ADL-OXB-induced hyperlocomotion. These results indicate that the activation of the OX2 receptor is involved in orexin A-induced hyperlocomotion. On the other hand, SB 334867 had no effects on orexin A-induced hyperlocomotion at all tested doses. Our present results were a somewhat inconsistent with a previous report which showed that intracerebroventricular administration of an OX1 receptor antagonist, SB 408124 partially blocked the total behavioral activity caused by orexin A in rats [10]. This discrepancy might be due to the differences in the compound itself and the route of administration of OX1 receptor antagonist between the two studies. We administered SB 334867 intraperitoneally, while Samson et al. administered SB 408124 intracerebroventricularly [10]. Because SB 408124 poorly crosses the blood-brain barrier (brain-to-plasma ratio 0.02) compared to SB 334867 (brain-toplasma ratio 0.29) [13], SB 334867 is intensively used for systemic administration in in vivo experiments in contrast to the OX1 receptor antagonist, SB 408124, although both the compounds had comparable antagonistic activity for the OX1 receptor [13,14]. In the present study, we selected the doses of SB 334867 as 3 and 10 mg/kg. Previously, it was reported that SB 334867 achieved about 80–90% occupancy for OX1 receptor from 30 min to 2 h at a subcutaneous dose of 10 mg/kg using ex vivo occupancy study in rats [13]. Taken together this finding with the profile that SB 334867 is an OX1 receptor antagonist with only approximately 50-fold selectivity over OX2 receptor [13], SB 334867 may also block OX2 receptor in addition to OX1 receptor at higher doses than 10 mg/kg. However, because we did not examine occupancy for OX2 receptor at various doses of SB 334867 in the present study, we could not
Fig. 2. Effects of SB 334867 (A), EMPA (A), and OX1 and OX2 receptor antagonist (B) on orexin A-induced hyperlocomotion in rats. SB 334867 (3–10 mg/kg, ip) or EMPA (30– 100 mg/kg, ip) was administered 30 min before administration of orexin A (1 nmol/rat, icv). The OX1 and OX2 receptor antagonist (3–30 mg/kg, po) was administered 1 h before the administration of orexin A. Orexin A-induced hyperlocomotion was measured as the total activity during a 120 min observation period immediately after administration of orexin A. Data are expressed as the mean SEM of 6 animals. Sharps represent a significant difference from the vehicle + vehicle-treated group, ##p < 0.01 (Student’s t-test). Asterisks represent a significant difference from the vehicle + orexin A-treated group, *p < 0.05, **p < 0.01 (Dunnett’s test).
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assess whether higher doses of SB 334867 affect orexin A-induced hyperlocomoion by the blockade of OX2 receptor. Our results indicate that the activation of the OX1 receptor may not make a major contribution to orexin A-induced hyperlocomotion. However, further studies with other systemically available OX1 receptor antagonists with high selectivity over OX2 receptor will be necessary to clarify the role of OX1 receptor in orexin A-induced hyperlocomotion. In addition, the effects of an OX1 and OX2 receptor antagonist in orexin A-induced hyperlocomotion were tested. The OX1 and OX2 receptor antagonist as well as EMPA antagonized orexin A-induced hyperlocomotion in a dose-dependent manner. These results suggest that the blockade of the OX2 receptor may be mainly involved in the antagonistic effect of the OX1 and OX2 receptor antagonist against orexin A-induced hyperlocomotion. In summary, we demonstrated that an OX2 receptor antagonist but not an OX1 receptor antagonist attenuated the hyperlocomotion caused by orexin A in rats. These results suggest that orexin Ainduced hyperlocomotion is mainly mediated by the activation of the OX2 receptor.
[2]
[3]
[4]
[5]
[6] [7]
[8]
[9]
[10]
Conflict of interest [11]
None. Acknowledgments
[12]
We thank Natsuko Yuge and Tatsuya Hoshino for their assistance in the in vivo experiments.
[13]
References
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