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Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice
Peptides 30 (2009) 1153–1157
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Peptides journal homepage: www.elsevier.com/locate/peptides
Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice Ning Xu 1, Haiting Wang 1, Li Fan, Qiang Chen * Department of Biochemistry and Molecular Biology, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2008 Received in revised form 12 February 2009 Accepted 12 February 2009 Available online 24 February 2009
The effect of apelin-13 on pain modulation at the supraspinal level was investigated in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/ mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the APJ receptor antagonist apelin-13(F13A), indicating an APJ receptor-mediated mechanism. Furthermore, naloxone, b-funaltrexamine and naloxonazine, could reverse the analgesic effect. However, naltrindole or nor-binaltorphimine could not reverse the effect, suggesting that m opioid receptor (primarily m1 opioid receptor subtype) is involved in the analgesic response evoked by apelin-13. Moreover, i.c.v. administration of apelin-13 potentiated the analgesic effect induced by morphine (i.c.v., 5 mg/kg) and this potentiated effect can be also reversed by naloxone. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Apelin-13 Apelin-13(F13A) Opioid receptor Antinociception
1. Introduction APJ is a G protein-coupled receptor that was originally isolated from human genomic DNA [22], subsequently cloned in mice [5] and rat [4,14,21]. It shares the closest identity to the angiotensin II type 1 (AT1) receptor ranging from 40% to 50% in the hydrophobic transmembrane regions, but it does not bind to angiotensin II [22]. The endogenous ligand for the APJ receptor was first isolated from bovine stomach extracts [30] and named apelin (APJ endogenous ligand). It derives from a 77 amino acid precursor, preproapelin, which can be cleaved into several molecular forms including apelin-36 and apelin-13 in different tissues [14,15]. Apelin-13 was found to exhibit significantly higher activity at the receptor than apelin-36 [30]. To date, some physiological effects of apelin have been reported. Apelin-13 has been shown to promote the acidification rate and inhibit cAMP production in cells expressing APJ [30] and it was demonstrated to stimulate the proliferation of gastric cells and to increase the secretion of cholecystokinin from dispersed intestinal endocrine cells [32]. Intraperitoneal (i.p.) administration of apelin13 increases drinking behavior [16], whereas intracerebroventricular (i.c.v.) administration decreases drinking behavior in dehydrated animals [25]. It has also been implied that apelin-13 was involved in the regulation of blood pressure [27,31], vasopressin
* Corresponding author. Tel.: +86 931 8915316; fax: +86 931 8912428. E-mail address: [email protected] (Q. Chen). 1 Both authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.02.011
release [25,29] and modulation of immune response [3,13]. However, to our knowledge, there have been no published papers about the nociceptive effect of apelin to date. The apelinergic system is widely distributed in both central system and periphery, particularly in the heart, kidney, lung, and mammary gland [9,14–16,21,25]. In the CNS, the distribution of apelin and its receptors in the amygdala, hypothalamus, dorsal raphe nucleus (DRN) and spinal cord suggests a possible role for apelin in nociception. Thus, the present study aimed to: (1) evaluate the effects of i.c.v. administration of apelin-13 on pain modulation and morphine-induced analgesia, and (2) investigate the mechanisms involved in the effect. 2. Materials and methods All experiments were carried out according to protocols approved by the Ethics Committee of Animal Experiments at Lanzhou University and in accordance with guidelines from the China Council on Animal Care and the International Association for the Study of Pain Committee for Research and Ethical Issues. Every effort was made to minimize the numbers and any suffering of the animals used in the following experiments. 2.1. Animals Male Kunming mice (20 1.0 g) were supplied by the Animal center of Lanzhou University (Lanzhou, China). The animals were housed (5–6/cage) at room temperature of 22 1 8C and 50–60% relative humidity. The room light was on a 12/12 h reversed cycle
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(light on 8:30 a.m. to 8:30 p.m.). The animals were allowed to adapt to this environment for a period of 1 week before the experiments. Food and water were freely available. 2.2. Chemicals Apelin-13 and apelin-13(F13A) were synthesized manually by the solid-phase peptide synthesis method and purified by highperformance liquid chromatography (HPLC) as described in a previous report [6]. Morphine hydrochloride was the product of Shenyang First Pharmaceutical Factory, China. Naloxone hydrochloride dihydrate (Fluka), b-funaltrexamine hydrochloride (b-FNA, Sigma), naloxonazine dihydrochloride (Sigma), naltrindole hydrochloride (NTI, Sigma) and nor-binaltorphimine dihydrochloride (nor-BNI, Sigma) are opioid receptor antagonists. Preparation of all stock solutions (except b-FNA) and their subsequent dilutions were performed in normal saline. Stock solutions were stored frozen in aliquots, thawed and diluted daily. The b-FNA aqueous solution was used promptly because of its instability. 2.3. Intracerebroventricular injection Intracerebroventricular administration was performed following the method described by Haley and McCormick [10]. The injection site was 1.5 mm from the midline, 1 mm from the bregma and 3 mm from the surface of the skull. Drugs were administered in a volume of 4 ml at a constant rate of 10 ml/min using a 25 ml Hamilton microsyringe. The proper injection site was verified in pilot experiments by administration and localization of methylene blue dye. 2.4. Nociceptive test The nociceptive response was assessed with 48.5 8C warm-water tail immersion test, using the method described by Fu et al. [7]. Every mouse was used only once. Briefly, the animals were gently restrained by hand, and their tails were immersed in a constanttemperature water bath set at 48.5 8C. The time elapsed prior to removal of the tail from the water surface was taken as the tail withdrawal latency (TWL). Every mouse was first tested for latency by immersing its tail in the water and recording the response time. Only those mice with the baseline latency within the range of 2.5– 4.5 s were selected for further studies, and a cut-off latency was set at 15 s to avoid damage to the tail. Before each drug trial, a series of six sequential predrug administration latency measurements were made to establish a stable baseline, each with a 10 min interval. The latencies of the last four tests were averaged to provide a control value. Typically, these values varied by <10%. Post-drug latency measurements were performed at 10, 15, 20, 30, 40, 50 and 60 min. To investigate the participation of the APJ receptor on the antinociceptive effect of apelin-13, the specific APJ receptor antagonist apelin-13(F13A) was co-injected with apelin-13. In order to further investigate the nociceptive mechanisms elicited by i.c.v. administration of apelin-13, classical opioid receptor antagonist naloxone was co-administrated with apelin13. To determine the type of opioid receptor involved in the antinociceptive effect of apelin-13, b-funaltrexamine hydrochloride, naloxonazine dihydrochloride, naltrindole hydrochloride or nor-binaltorphimine dihydrochloride (nor-BNI), m-, m1 , d-, kopioid receptor antagonists, respectively, were used. 2.5. Statistical analysis The data were calculated as the percentage change of tail withdrawal latency from the baseline level according to the
formula: percentage change of TWL = [(post-drug latency predrug latency)/predrug latency] 100. The results were expressed as means S.E.M. Each group consisted of 8–12 mice. Data were analyzed using one-way analyses of ANOVA followed by the Dunnett’s post hoc comparisons on all time course studies. A value of P < 0.05 was selected as indicative of a significant difference. 3. Results 3.1. In vivo effect of apelin-13 on the nociception after i.c.v. administration Fig. 1 illustrates the dose- and time-related analgesic effect of i.c.v. administration of apelin-13 in 48.5 8C warm-water tail immersion test in conscious mice. The i.c.v. administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/mouse) produced a significant dose-related increase in tail withdrawal latencies. This effect reached a maximum at 15 min and terminated about 40 min after i.c.v. injection. The percent change of TWL at 15 min after i.c.v. administration of 0.3, 0.5, 0.8 and 3 mg/mouse apelin-13 was 33.85 4.13%(8), 41.21 3.15%(12), 57.17 3.57%(10) and 28.66 3.02%(11), respectively (vs. NS group, each P < 0.001). The analgesic effect was evoked most effectively by 0.8 mg apelin-13, and there was no further increase of analgesic behavior by either higher or lower dose of apelin-13. 3.2. Effect of the APJ receptor antagonist apelin-13(F13A) on the analgesia induced by apelin-13 To explore whether the APJ receptor is involved in apelin-13induced analgesic responses, the APJ receptor antagonist apelin13(F13A) was co-injected with apelin-13. The result showed that apelin-13(F13A) fully blocked the apelin-13-elicited antinociceptive effect in the tail immersion test (Fig. 2), indicating that the antinociceptive effect of the i.c.v. administration of apelin-13 is mediated through the activation of the APJ receptor. 3.3. Effects of opioid receptor antagonists on the analgesia induced by apelin-13 Naloxone, a broad-spectrum opioid receptor antagonist, fully abolished the analgesic effect of apelin-13, indicating that opioid receptors were involved in the antinociceptive effect of apelin-13 (Fig. 3A). To further investigate which opioid receptors are involved in the response, the effects of b-FNA (an irreversible m
Fig. 1. Dose- and time-related analgesic effect of i.c.v. administration of apelin-13 in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 8–12/group. Post hoc analysis for the duration of response after i.c.v. administration of apelin-13 groups indicated significant difference from the group of i.c.v. administration of normal saline (NS) on appropriate time (**P < 0.01, ***P < 0.001). AP13, apelin-13; NS, normal saline.
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Fig. 2. The effect of i.c.v. administration of APJ receptor antagonist apelin-13(F13A) on i.c.v. apelin-13 induced analgesia in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9– 12/group. Post hoc analysis for the duration of response after i.c.v. co-administration of apelin-13(F13A) and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (*P < 0.05, ***P < 0.001). AP13, apelin-13; AP13A, apelin-13(F13A).
opioid receptor antagonist), naloxonazine (an irreversible m1 opioid receptor antagonist), NTI (a d opioid receptor antagonist) and nor-BNI (a high selective k opioid receptor antagonist) were determined. Both b-FNA and naloxonazine significantly abolished the analgesic response induced by apelin-13 (Figs. 3A and 4), while the analgesic response induced by apelin-13 was not significantly modified by NTI or nor-BNI, indicating that only m opioid receptor (primary m1 subtype) is involved in the analgesic response evoked by apelin-13. It should be noted that the time and dose of NTI or nor-BNI we chosen were based on previous studies [1,7,12,19,28]. 3.4. Effect of apelin-13 (i.c.v.) on analgesia elicited by morphine (i.c.v.) Fig. 5A shows that the co-administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/mouse) and morphine (5 mg/kg) potentiated the analgesic effect elicited by morphine. Co-administration of apeline-13 of 0.8 mg/mouse with morphine (5 mg/kg) produced a change of TWL to 91.97 4.84% compared with 40.69 3.19% of morphine alone observed at 15 min (Fig. 6). The potentiated analgesic response was completely blocked by naloxone.
Fig. 3. (A) The effect of i.c.v. administration of various opioid receptors antagonists on apelin-13 induced analgesia in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9–11/ group. Post hoc analysis for the duration of response after i.c.v. co-administration of various opioid receptor antagonist and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (**P < 0.01, ***P < 0.001). AP13, apelin-13; b-FNA, b-funaltrexamine; NLXZ, naloxonazine; NTI, naltrindole; nor-BNI, nor-binaltorphimine. (B) The effect of i.c.v. administration of opioid receptor antagonists alone in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9–11/group. Post hoc analysis for the duration of response after i.c.v. administration of all antagonists groups did not indicate a significant difference from NS group at appropriate times.
4. Discussion The study presents evidence that apelin-13, a novel neuropeptide, plays a significant role in the modulation of pain response at the supraspinal level in mice. Our results also suggest that the analgesic effect of apelin-13 was mediated by the activation of the APJ receptor and endogenous opioid system. The structure and anatomical distribution similarities between APJ/apelin and the AT1/angiotensin II may provide clues about the physiological functions of the apelin system. Antinociception following i.c.v. administration of angiotensin II has been proved in several rodent pain models [8,11,24]. Pelegrini-dasilva et al. found that, after injection into the periaqueductal gray matter, angiotensin II produced analgesic effect in the tail immersion test and its effect was significantly antagonized by AT1 and AT2 antagonists [23]. In the present study, we also found that i.c.v. administration of apelin-13 (0.3–3 mg/mouse) produced a dose- and time-related analgesic effect, evoked most effectively by a 0.8 mg/mouse dosage. The ‘‘U-shaped’’ dose response relationships might represent situations when apelin-13 is an agonist at low concentrations but displays partial reversal of agonistic activity at higher doses. Another possibility is that apelin-13 may coactivate receptors other than classical opioid
Fig. 4. The effect of various opioid receptors antagonists on apelin-13 induced analgesia at 15 min after i.c.v. administration in 48.5 8C warm-water tail immersion test in mice. All data at each dose point are presented as % change of TFL S.E.M. for n = 9–11/group. Post hoc analysis for the duration of response at 15 min after i.c.v. coadministration of various opioid receptor antagonist and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (***P < 0.001). S, saline; AP13, apelin-13; b-FNA, b-funaltrexamine; NLXZ, naloxonazine; NTI, naltrindole; nor-BNI, nor-binaltorphimine.
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Fig. 5. (A) Dose- and time-related synergistic analgesic effects of the i.c.v. coadministration of apelin-13 and MRP in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9– 11/group. The statistical significance of differences between the groups was assessed with a one-way analysis of variance (ANOVA) followed by the Dunnett’s test. *P < 0.05, **P < 0.01 and ***P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual MRP. AP13, apelin-13; MRP, morphine. (B) Reversing effect of i.c.v. administration of naloxone on analgesia of co-administration of morphine and apelin13 in 48.5 8C warm-water tail immersion test in mice. All data at each time point are showed as % change of TFL S.E.M. for n = 9–11/group. **P < 0.01, ***P < 0.001, statistically significant differences in comparison with apelin-13 plus morphine.
Fig. 6. Dose-related synergistic analgesic effects of apelin-13 and MRP at 15 min after i.c.v. co-administration in 48.5 8C warm-water tail immersion test in mice. All data at each dose point are presented as % change of TFL S.E.M. for n = 9–11/group. The statistical significance of differences between the groups was assessed with a oneway analysis of variance (ANOVA) followed by the Dunnett’s test. **P < 0.01 and ***P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual MRP. ##P < 0.01 and ###P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual apelin-13. AP13, apelin-13 and MRP, morphine.
receptors and this compromises its antinociceptive efficacy. Some reports about similar phenomenons show interactions with different receptor systems [18]. To our knowledge, this is the first report showing that apelin-13 affects pain modulation. This result is consistent with the anatomical distribution of apelin receptors that have been detected in the spinal cord, hypothalamus, dorsal raphe nucleus and medulla oblongata [4,14,20]. Apelin-13(F13A) was first reported by Lee et al. as a specific antagonist of the APJ receptor and can block hypotensive effects induced by apelin-13 [17]. Thus, the result of present study that apelin-13(F13A) can block the antinociceptive effect of apelin-13 suggests that the analgesic effect of apelin-13 is mediated through the APJ receptor. Befort et al. found that apelin transcript levels were reduced in the lateral hypothalamus of morphine-dependent mice, suggesting a potential role for apelin in opioid signaling [2]. In the present study, we found that i.c.v. treatment with naloxone, a broad-spectrum opioid receptor antagonist, completely reversed the antinociceptive effect induced by apelin-13. This indicated that the opioid system was involved in the analgesic effect of apelin-13. Considering the fact that the antinociceptive effect of i.c.v. administered apelin-13 was sensitive to naloxone or apelin13(F13A), we proposed that i.c.v. administration of apelin-13 may activate the APJ receptor first, then apelin-13 secondarily induce the release of endogenous opioid peptides, thereby producing opioid-dependent analgesia. In support of this, anatomic evidence showed localization of apelin in opioid rich brain areas of the arcuate nucleus of hypothalamus (ARC) and spinal trigeminal nucleus [26]. Thus, our findings may provide an indirect functional interrelationship between apelin-13 and opioid systems. The type of opioid receptor involved in the antinociceptive effect of apelin-13 was also determined. We found that both b-FNA and naloxonazine significantly abolish the antinociceptive effect induced by apelin-13 in the tail immersion test. This provides further evidence that opioid receptors were involved in the antinociceptive effect of apelin-13. In contrast, administration of the selective k or d-receptor antagonist did not significantly affect the antinociceptive response exerted by apelin-13. These data indicate that apelin-13 inhibits nociception by activating m opioid receptors (primary m1 opioid receptor subtype). This finding is consistent with a previous report that is also suggesting a potential interaction between apelin and m opioid receptors [2]. In that study it was shown that apelin transcript levels were reduced as a consequence of m opioid receptor stimulation. In the present study, we also assessed the effect of apelin-13 on morphine-induced analgesia. We found that (1) i.c.v. administration of apelin-13 could significantly potentiate the antinociceptive effects of morphine, suggesting that apelin-13 may have a modulatory effect on opioid (morphine) analgesia and (2) the opioid receptor antagonist naloxone could block the potentiated analgesic response effectively. Consistent with our hypothesis, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of apelin-13 to release endogenous opioid peptides. The synergistic analgesic relationship of morphine and apelin-13 support the hypothesis that supraspinal apelin and opioid systems have a functional interaction in the modulation of nociceptive responses. In summary, data from our present experiments strongly indicates, for the first time, that i.c.v. administration of apelin-13 can induce a dose- and time-related analgesic effect in mice. The effect was mediated through the activation of APJ receptors firstly, and then by exciting m opioid receptors (primary m1 opioid receptors subtype) in opioidergic neurons. We also found that apelin-13 can potentiate the antinociceptive effect of morphine through the opioid system. These results should facilitate the
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analysis of the role of apelin-13 in acute pain and may open novel pharmacological interventions. Acknowledgments This study was supported by grants from the National Nature Science Foundation of China (No. J0630644). The authors would like to thank the other members of the group for their suggestions and help in the research.
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Supraspinal administration of apelin-13 induces antinociception via the opioid receptor in mice Ning Xu 1, Haiting Wang 1, Li Fan, Qiang Chen * Department of Biochemistry and Molecular Biology, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2008 Received in revised form 12 February 2009 Accepted 12 February 2009 Available online 24 February 2009
The effect of apelin-13 on pain modulation at the supraspinal level was investigated in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/ mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the APJ receptor antagonist apelin-13(F13A), indicating an APJ receptor-mediated mechanism. Furthermore, naloxone, b-funaltrexamine and naloxonazine, could reverse the analgesic effect. However, naltrindole or nor-binaltorphimine could not reverse the effect, suggesting that m opioid receptor (primarily m1 opioid receptor subtype) is involved in the analgesic response evoked by apelin-13. Moreover, i.c.v. administration of apelin-13 potentiated the analgesic effect induced by morphine (i.c.v., 5 mg/kg) and this potentiated effect can be also reversed by naloxone. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Apelin-13 Apelin-13(F13A) Opioid receptor Antinociception
1. Introduction APJ is a G protein-coupled receptor that was originally isolated from human genomic DNA [22], subsequently cloned in mice [5] and rat [4,14,21]. It shares the closest identity to the angiotensin II type 1 (AT1) receptor ranging from 40% to 50% in the hydrophobic transmembrane regions, but it does not bind to angiotensin II [22]. The endogenous ligand for the APJ receptor was first isolated from bovine stomach extracts [30] and named apelin (APJ endogenous ligand). It derives from a 77 amino acid precursor, preproapelin, which can be cleaved into several molecular forms including apelin-36 and apelin-13 in different tissues [14,15]. Apelin-13 was found to exhibit significantly higher activity at the receptor than apelin-36 [30]. To date, some physiological effects of apelin have been reported. Apelin-13 has been shown to promote the acidification rate and inhibit cAMP production in cells expressing APJ [30] and it was demonstrated to stimulate the proliferation of gastric cells and to increase the secretion of cholecystokinin from dispersed intestinal endocrine cells [32]. Intraperitoneal (i.p.) administration of apelin13 increases drinking behavior [16], whereas intracerebroventricular (i.c.v.) administration decreases drinking behavior in dehydrated animals [25]. It has also been implied that apelin-13 was involved in the regulation of blood pressure [27,31], vasopressin
* Corresponding author. Tel.: +86 931 8915316; fax: +86 931 8912428. E-mail address: [email protected] (Q. Chen). 1 Both authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.02.011
release [25,29] and modulation of immune response [3,13]. However, to our knowledge, there have been no published papers about the nociceptive effect of apelin to date. The apelinergic system is widely distributed in both central system and periphery, particularly in the heart, kidney, lung, and mammary gland [9,14–16,21,25]. In the CNS, the distribution of apelin and its receptors in the amygdala, hypothalamus, dorsal raphe nucleus (DRN) and spinal cord suggests a possible role for apelin in nociception. Thus, the present study aimed to: (1) evaluate the effects of i.c.v. administration of apelin-13 on pain modulation and morphine-induced analgesia, and (2) investigate the mechanisms involved in the effect. 2. Materials and methods All experiments were carried out according to protocols approved by the Ethics Committee of Animal Experiments at Lanzhou University and in accordance with guidelines from the China Council on Animal Care and the International Association for the Study of Pain Committee for Research and Ethical Issues. Every effort was made to minimize the numbers and any suffering of the animals used in the following experiments. 2.1. Animals Male Kunming mice (20 1.0 g) were supplied by the Animal center of Lanzhou University (Lanzhou, China). The animals were housed (5–6/cage) at room temperature of 22 1 8C and 50–60% relative humidity. The room light was on a 12/12 h reversed cycle
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(light on 8:30 a.m. to 8:30 p.m.). The animals were allowed to adapt to this environment for a period of 1 week before the experiments. Food and water were freely available. 2.2. Chemicals Apelin-13 and apelin-13(F13A) were synthesized manually by the solid-phase peptide synthesis method and purified by highperformance liquid chromatography (HPLC) as described in a previous report [6]. Morphine hydrochloride was the product of Shenyang First Pharmaceutical Factory, China. Naloxone hydrochloride dihydrate (Fluka), b-funaltrexamine hydrochloride (b-FNA, Sigma), naloxonazine dihydrochloride (Sigma), naltrindole hydrochloride (NTI, Sigma) and nor-binaltorphimine dihydrochloride (nor-BNI, Sigma) are opioid receptor antagonists. Preparation of all stock solutions (except b-FNA) and their subsequent dilutions were performed in normal saline. Stock solutions were stored frozen in aliquots, thawed and diluted daily. The b-FNA aqueous solution was used promptly because of its instability. 2.3. Intracerebroventricular injection Intracerebroventricular administration was performed following the method described by Haley and McCormick [10]. The injection site was 1.5 mm from the midline, 1 mm from the bregma and 3 mm from the surface of the skull. Drugs were administered in a volume of 4 ml at a constant rate of 10 ml/min using a 25 ml Hamilton microsyringe. The proper injection site was verified in pilot experiments by administration and localization of methylene blue dye. 2.4. Nociceptive test The nociceptive response was assessed with 48.5 8C warm-water tail immersion test, using the method described by Fu et al. [7]. Every mouse was used only once. Briefly, the animals were gently restrained by hand, and their tails were immersed in a constanttemperature water bath set at 48.5 8C. The time elapsed prior to removal of the tail from the water surface was taken as the tail withdrawal latency (TWL). Every mouse was first tested for latency by immersing its tail in the water and recording the response time. Only those mice with the baseline latency within the range of 2.5– 4.5 s were selected for further studies, and a cut-off latency was set at 15 s to avoid damage to the tail. Before each drug trial, a series of six sequential predrug administration latency measurements were made to establish a stable baseline, each with a 10 min interval. The latencies of the last four tests were averaged to provide a control value. Typically, these values varied by <10%. Post-drug latency measurements were performed at 10, 15, 20, 30, 40, 50 and 60 min. To investigate the participation of the APJ receptor on the antinociceptive effect of apelin-13, the specific APJ receptor antagonist apelin-13(F13A) was co-injected with apelin-13. In order to further investigate the nociceptive mechanisms elicited by i.c.v. administration of apelin-13, classical opioid receptor antagonist naloxone was co-administrated with apelin13. To determine the type of opioid receptor involved in the antinociceptive effect of apelin-13, b-funaltrexamine hydrochloride, naloxonazine dihydrochloride, naltrindole hydrochloride or nor-binaltorphimine dihydrochloride (nor-BNI), m-, m1 , d-, kopioid receptor antagonists, respectively, were used. 2.5. Statistical analysis The data were calculated as the percentage change of tail withdrawal latency from the baseline level according to the
formula: percentage change of TWL = [(post-drug latency predrug latency)/predrug latency] 100. The results were expressed as means S.E.M. Each group consisted of 8–12 mice. Data were analyzed using one-way analyses of ANOVA followed by the Dunnett’s post hoc comparisons on all time course studies. A value of P < 0.05 was selected as indicative of a significant difference. 3. Results 3.1. In vivo effect of apelin-13 on the nociception after i.c.v. administration Fig. 1 illustrates the dose- and time-related analgesic effect of i.c.v. administration of apelin-13 in 48.5 8C warm-water tail immersion test in conscious mice. The i.c.v. administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/mouse) produced a significant dose-related increase in tail withdrawal latencies. This effect reached a maximum at 15 min and terminated about 40 min after i.c.v. injection. The percent change of TWL at 15 min after i.c.v. administration of 0.3, 0.5, 0.8 and 3 mg/mouse apelin-13 was 33.85 4.13%(8), 41.21 3.15%(12), 57.17 3.57%(10) and 28.66 3.02%(11), respectively (vs. NS group, each P < 0.001). The analgesic effect was evoked most effectively by 0.8 mg apelin-13, and there was no further increase of analgesic behavior by either higher or lower dose of apelin-13. 3.2. Effect of the APJ receptor antagonist apelin-13(F13A) on the analgesia induced by apelin-13 To explore whether the APJ receptor is involved in apelin-13induced analgesic responses, the APJ receptor antagonist apelin13(F13A) was co-injected with apelin-13. The result showed that apelin-13(F13A) fully blocked the apelin-13-elicited antinociceptive effect in the tail immersion test (Fig. 2), indicating that the antinociceptive effect of the i.c.v. administration of apelin-13 is mediated through the activation of the APJ receptor. 3.3. Effects of opioid receptor antagonists on the analgesia induced by apelin-13 Naloxone, a broad-spectrum opioid receptor antagonist, fully abolished the analgesic effect of apelin-13, indicating that opioid receptors were involved in the antinociceptive effect of apelin-13 (Fig. 3A). To further investigate which opioid receptors are involved in the response, the effects of b-FNA (an irreversible m
Fig. 1. Dose- and time-related analgesic effect of i.c.v. administration of apelin-13 in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 8–12/group. Post hoc analysis for the duration of response after i.c.v. administration of apelin-13 groups indicated significant difference from the group of i.c.v. administration of normal saline (NS) on appropriate time (**P < 0.01, ***P < 0.001). AP13, apelin-13; NS, normal saline.
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Fig. 2. The effect of i.c.v. administration of APJ receptor antagonist apelin-13(F13A) on i.c.v. apelin-13 induced analgesia in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9– 12/group. Post hoc analysis for the duration of response after i.c.v. co-administration of apelin-13(F13A) and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (*P < 0.05, ***P < 0.001). AP13, apelin-13; AP13A, apelin-13(F13A).
opioid receptor antagonist), naloxonazine (an irreversible m1 opioid receptor antagonist), NTI (a d opioid receptor antagonist) and nor-BNI (a high selective k opioid receptor antagonist) were determined. Both b-FNA and naloxonazine significantly abolished the analgesic response induced by apelin-13 (Figs. 3A and 4), while the analgesic response induced by apelin-13 was not significantly modified by NTI or nor-BNI, indicating that only m opioid receptor (primary m1 subtype) is involved in the analgesic response evoked by apelin-13. It should be noted that the time and dose of NTI or nor-BNI we chosen were based on previous studies [1,7,12,19,28]. 3.4. Effect of apelin-13 (i.c.v.) on analgesia elicited by morphine (i.c.v.) Fig. 5A shows that the co-administration of apelin-13 (0.3, 0.5, 0.8 and 3 mg/mouse) and morphine (5 mg/kg) potentiated the analgesic effect elicited by morphine. Co-administration of apeline-13 of 0.8 mg/mouse with morphine (5 mg/kg) produced a change of TWL to 91.97 4.84% compared with 40.69 3.19% of morphine alone observed at 15 min (Fig. 6). The potentiated analgesic response was completely blocked by naloxone.
Fig. 3. (A) The effect of i.c.v. administration of various opioid receptors antagonists on apelin-13 induced analgesia in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9–11/ group. Post hoc analysis for the duration of response after i.c.v. co-administration of various opioid receptor antagonist and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (**P < 0.01, ***P < 0.001). AP13, apelin-13; b-FNA, b-funaltrexamine; NLXZ, naloxonazine; NTI, naltrindole; nor-BNI, nor-binaltorphimine. (B) The effect of i.c.v. administration of opioid receptor antagonists alone in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9–11/group. Post hoc analysis for the duration of response after i.c.v. administration of all antagonists groups did not indicate a significant difference from NS group at appropriate times.
4. Discussion The study presents evidence that apelin-13, a novel neuropeptide, plays a significant role in the modulation of pain response at the supraspinal level in mice. Our results also suggest that the analgesic effect of apelin-13 was mediated by the activation of the APJ receptor and endogenous opioid system. The structure and anatomical distribution similarities between APJ/apelin and the AT1/angiotensin II may provide clues about the physiological functions of the apelin system. Antinociception following i.c.v. administration of angiotensin II has been proved in several rodent pain models [8,11,24]. Pelegrini-dasilva et al. found that, after injection into the periaqueductal gray matter, angiotensin II produced analgesic effect in the tail immersion test and its effect was significantly antagonized by AT1 and AT2 antagonists [23]. In the present study, we also found that i.c.v. administration of apelin-13 (0.3–3 mg/mouse) produced a dose- and time-related analgesic effect, evoked most effectively by a 0.8 mg/mouse dosage. The ‘‘U-shaped’’ dose response relationships might represent situations when apelin-13 is an agonist at low concentrations but displays partial reversal of agonistic activity at higher doses. Another possibility is that apelin-13 may coactivate receptors other than classical opioid
Fig. 4. The effect of various opioid receptors antagonists on apelin-13 induced analgesia at 15 min after i.c.v. administration in 48.5 8C warm-water tail immersion test in mice. All data at each dose point are presented as % change of TFL S.E.M. for n = 9–11/group. Post hoc analysis for the duration of response at 15 min after i.c.v. coadministration of various opioid receptor antagonist and apelin-13 groups indicated significant difference from i.c.v. administration of apelin-13 groups on appropriate time (***P < 0.001). S, saline; AP13, apelin-13; b-FNA, b-funaltrexamine; NLXZ, naloxonazine; NTI, naltrindole; nor-BNI, nor-binaltorphimine.
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Fig. 5. (A) Dose- and time-related synergistic analgesic effects of the i.c.v. coadministration of apelin-13 and MRP in 48.5 8C warm-water tail immersion test in mice. All data at each time point are presented as % change of TFL S.E.M. for n = 9– 11/group. The statistical significance of differences between the groups was assessed with a one-way analysis of variance (ANOVA) followed by the Dunnett’s test. *P < 0.05, **P < 0.01 and ***P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual MRP. AP13, apelin-13; MRP, morphine. (B) Reversing effect of i.c.v. administration of naloxone on analgesia of co-administration of morphine and apelin13 in 48.5 8C warm-water tail immersion test in mice. All data at each time point are showed as % change of TFL S.E.M. for n = 9–11/group. **P < 0.01, ***P < 0.001, statistically significant differences in comparison with apelin-13 plus morphine.
Fig. 6. Dose-related synergistic analgesic effects of apelin-13 and MRP at 15 min after i.c.v. co-administration in 48.5 8C warm-water tail immersion test in mice. All data at each dose point are presented as % change of TFL S.E.M. for n = 9–11/group. The statistical significance of differences between the groups was assessed with a oneway analysis of variance (ANOVA) followed by the Dunnett’s test. **P < 0.01 and ***P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual MRP. ##P < 0.01 and ###P < 0.001, statistically significant differences between apelin-13 plus MRP vs. individual apelin-13. AP13, apelin-13 and MRP, morphine.
receptors and this compromises its antinociceptive efficacy. Some reports about similar phenomenons show interactions with different receptor systems [18]. To our knowledge, this is the first report showing that apelin-13 affects pain modulation. This result is consistent with the anatomical distribution of apelin receptors that have been detected in the spinal cord, hypothalamus, dorsal raphe nucleus and medulla oblongata [4,14,20]. Apelin-13(F13A) was first reported by Lee et al. as a specific antagonist of the APJ receptor and can block hypotensive effects induced by apelin-13 [17]. Thus, the result of present study that apelin-13(F13A) can block the antinociceptive effect of apelin-13 suggests that the analgesic effect of apelin-13 is mediated through the APJ receptor. Befort et al. found that apelin transcript levels were reduced in the lateral hypothalamus of morphine-dependent mice, suggesting a potential role for apelin in opioid signaling [2]. In the present study, we found that i.c.v. treatment with naloxone, a broad-spectrum opioid receptor antagonist, completely reversed the antinociceptive effect induced by apelin-13. This indicated that the opioid system was involved in the analgesic effect of apelin-13. Considering the fact that the antinociceptive effect of i.c.v. administered apelin-13 was sensitive to naloxone or apelin13(F13A), we proposed that i.c.v. administration of apelin-13 may activate the APJ receptor first, then apelin-13 secondarily induce the release of endogenous opioid peptides, thereby producing opioid-dependent analgesia. In support of this, anatomic evidence showed localization of apelin in opioid rich brain areas of the arcuate nucleus of hypothalamus (ARC) and spinal trigeminal nucleus [26]. Thus, our findings may provide an indirect functional interrelationship between apelin-13 and opioid systems. The type of opioid receptor involved in the antinociceptive effect of apelin-13 was also determined. We found that both b-FNA and naloxonazine significantly abolish the antinociceptive effect induced by apelin-13 in the tail immersion test. This provides further evidence that opioid receptors were involved in the antinociceptive effect of apelin-13. In contrast, administration of the selective k or d-receptor antagonist did not significantly affect the antinociceptive response exerted by apelin-13. These data indicate that apelin-13 inhibits nociception by activating m opioid receptors (primary m1 opioid receptor subtype). This finding is consistent with a previous report that is also suggesting a potential interaction between apelin and m opioid receptors [2]. In that study it was shown that apelin transcript levels were reduced as a consequence of m opioid receptor stimulation. In the present study, we also assessed the effect of apelin-13 on morphine-induced analgesia. We found that (1) i.c.v. administration of apelin-13 could significantly potentiate the antinociceptive effects of morphine, suggesting that apelin-13 may have a modulatory effect on opioid (morphine) analgesia and (2) the opioid receptor antagonist naloxone could block the potentiated analgesic response effectively. Consistent with our hypothesis, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of apelin-13 to release endogenous opioid peptides. The synergistic analgesic relationship of morphine and apelin-13 support the hypothesis that supraspinal apelin and opioid systems have a functional interaction in the modulation of nociceptive responses. In summary, data from our present experiments strongly indicates, for the first time, that i.c.v. administration of apelin-13 can induce a dose- and time-related analgesic effect in mice. The effect was mediated through the activation of APJ receptors firstly, and then by exciting m opioid receptors (primary m1 opioid receptors subtype) in opioidergic neurons. We also found that apelin-13 can potentiate the antinociceptive effect of morphine through the opioid system. These results should facilitate the
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analysis of the role of apelin-13 in acute pain and may open novel pharmacological interventions. Acknowledgments This study was supported by grants from the National Nature Science Foundation of China (No. J0630644). The authors would like to thank the other members of the group for their suggestions and help in the research.
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