Antidepressant-Like Effects of Intracerebroventricular Injection of Nociceptin Analogs in Mice

Abstract

Opioid receptors and their endogenous ligands are novel targets for the treatment of depression. The nociception (NOP) receptor is structurally similar to the opioid receptor, but NOP is known to have a low affinity for the opioid receptor subtypes μ, δ, and κ. In previous studies, we synthesized peptides with a high affinity for opioid receptors and investigated their antidepressant-like effects in mice. However, we have not yet examined whether NOP-related analogs have antidepressant-like effects. Herein, we synthesized NOP analogs (peptide-1–peptide-8) by solid-phase peptide synthesis using the 9-fluorenylmethyloxycarbony (Fmoc) method with Acetyl-Arg-Tyr-Tyr-Arg-Ile-Arg-NH₂ (Ac-RYYRIR-NH₂) as the lead compound. We examined the affinities and antagonistic activities of the analogs for the NOP receptor using receptor-binding and mouse vas deferens assays, and their effects on the duration of immobile behavior in a tail suspension test. Peptide-6 showed a high affinity and antagonistic activity for the NOP receptor. The intracerebroventricular administration of peptide-6 in mice shortened the duration of immobile behavior, whereas the co-administration of NOP inhibited this effect. Moreover, intracerebroventricular administration of the selective NOP receptor antagonist J-113397 showed antidepressant-like effects in mice. These data suggest that peptide-6 exerts an antidepressant-like effect via inactivation of the central NOP receptor in mice and may represent a lead compound for the development of antidepressant drugs in the future.

INTRODUCTION

Nociceptin (NOP) and its receptors are new targets for the treatment of depression.¹⁾ Although NOP receptors have structural similarity to opioid receptors, NOP has low affinity for the μ, δ, and κ opioid subtype receptors.²⁾ Furthermore, NOP receptors are widely expressed in several brain regions, including the cortex, hippocampus, amygdala, dorsal tegmental nucleus, and those associated with mood disorders.³⁾ NOP is involved in a variety of physiological and motivational processes, including learning and memory, pain, anxiety, stress, depression, addiction, and eating behavior, consistent with its anatomic localization in the brain.⁴⁾ Peripherally, NOP produces vasodilatory, inotropic, gastroprokinetic, inflammatory, and antitussive effects.^4–7) Although the stimulation of typical opioid receptors has been reported to produce antidepressant-like effects, stimulated NOP receptors induce depression-like behaviors.^8–10) In addition, inhibition of NOP receptors has been reported to exert antidepressant-like effects.⁸⁾ Therefore, NOP receptor antagonists are expected to exert antidepressant-like effects.

Intermediate molecule drugs, such as peptides and nucleic acids, and consisting of molecular weights ranging from 500 to 10000 are positioned between small-molecule and high-molecule drugs. They generally exhibit fewer side effects than small-molecule drugs and higher intracellular translocation than high-molecule drugs.^11,12) Intermediate molecule drugs are expected to become a leading modality for the next generation as the number of drug targets for conventional pharmaceuticals is decreasing. Therefore, we have been studying the bioactivity of peptide compounds for drug discovery research applications.

Acetyl-Arg-Tyr-Tyr-Arg-Ile-Arg-NH₂ (Ac-RYYRIR-NH₂) is a short peptide with high affinity for NOP receptors, producing an antagonistic effect when bound.¹³⁾ The N-terminal 1–5 residues are essential for receptor affinity, and the 5th residue significantly affects agonist or antagonist action.¹⁴⁾ Amino acid residues with l-type fatty alkyl side chains have high antagonistic activity, whereas d-type amino acid residues exhibit only agonist action.¹⁴⁾ In this study, eight NOP analogs (peptide-1–peptide-8) were prepared by solid-phase peptide synthesis using the 9-fluorenylmethyloxycarbony (Fmoc) method, with Ac-RYYRIR-NH₂ as the lead compound. However, whether these analogs exert antidepressant-like effects in rodents remains unknown.

Consequently, we examined the affinities and pA₂ values of the resulting analogs for NOP receptors, their effects on the duration of immobility behavior, and the mechanisms behind their antidepressant-like effects.

MATERIALS AND METHODS

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tohoku Medical and Pharmaceutical University (Approval No.: A23061-cn) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize suffering and to reduce the number of animals used.

Animals

Male ddY mice (28–32 g; Japan SLC, Shizuoka, Japan) were used in all experiments (n = 533 mice). The mice were housed in cages with free access to food and water under controlled temperature (22 ± 2°C) and humidity (55 ± 5%) conditions on a 12-h light–dark cycle (lights on: 07:00 to 19:00).

Drugs

NOP analogs, peptide-1–peptide-8, were synthesized in-house. The amino acid sequences of these analogs are listed in Table 1. NOP analogs and NOP (Peptide Institute, Osaka, Japan) were dissolved in Ringer’s solution, whereas J-113397 was dissolved in Ringer’s solution containing 15% dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Intracerebroventricular injection was performed as previously described.^15,16) To prevent the head of the mouse from moving, the animal was firmly secured to the bench by grasping the loose skin on the back of the head with the thumb and index finger and stretching the skin on both sides of the neck. Some of the compounds (5 µL) were then administered using a 50 µL Hamilton microsyringe attached to a 27G disposable needle, stabbed vertically from the skull into the left lateral ventricle of unanesthetized mice, and administered at a constant rate of 1 µL/2 s. Naloxone (NAL; Sigma-Aldrich, St. Louis, MO, U.S.A.) was dissolved in saline and intraperitoneally administered at a dose of 0.1 mL/10 g of body weight. The doses of J-113397, NOP, and NAL used in this study were based on previous studies.^9,17) Behavioral tests were carried out 30 min after drug treatment.

Table 1. NOP Receptor Binding Affinity and pA₂ Values for Peptides 1–8

N.D., Not Determined.

Receptor Binding Assay

The affinity of each peptide for the NOP receptor was assessed using a binding assay of HEK 293 cells expressing the human NOP receptor, as previously described.¹⁴⁾ [³H]NOP binding was performed as follows: HEK 293 cell membranes (10.6 µg protein content), 0.2 nM [³H]NOP (5.62 TBq/mmol), and synthetic peptides in 50 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM MgCl₂ were incubated for 1 h at 25°C in siliconized tubes. After incubation, each reaction mixture was filtered through a glass filter (GF/B) and washed thrice with 2 mL of ice-cold HEPES buffer. The radioactivity bound to the filters was measured using a Beckman 9800 liquid scintillation counter (Beckman LS9800). The IC₅₀ values were determined from the log dose-response curves. The IC₅₀ values for each peptide at the NOP receptor were calculated as the average of results from four to six independently performed binding experiments. Except for peptide-6, these results have been previously published.¹⁴⁾

Mouse Vas Deferens (MVD) Assay

ddY mice weighing 25–30 g were used in this experiment. After cervical dislocation, the MVD was removed and mounted in a 10 mL organ bath containing Mg²⁺-free Krebs solution with 0.1 mM ascorbic acid and 0.027 mM EDTA 4Na, as described by Hughes et al.¹⁸⁾ The Krebs solution was maintained at 37°C and continuously perfused with a 95% O₂/5% CO₂. The tissue was stimulated transmurally with consecutive trains of rectilinear pulses delivered every 20 s, with each train consisting of seven 1-ms pulses spaced 10 ms apart. To assess the antagonistic potency of peptides 1, 3, and 6, the pA₂ value was determined by measuring the two-fold rightward shift in the dose–response curve of NOP. These peptides were added to the organ bath 15 min before the addition of NOP. Except for peptide-6, these results have been previously published.¹⁴⁾

Tail Suspension Test

The tail suspension test was performed as previously described^9,10) to evaluate the antidepressant-like effects of the NOP analogs. The mice were suspended with their tails taped 30 cm above the floor. An investigator blinded to the treatment assignments observed and recorded the immobility time for 10 min.

Locomotor Activity

Locomotor activity was determined using SUPERMEX software (Muromachi Kikai Co., Ltd., Tokyo, Japan). The details of the apparatus have been previously described.⁹⁾ Locomotor activity was measured for 10 min during the light phase, between 11:00 a.m. and 15:00 p.m. Each mouse was placed in a SUPERMEX activity box for 15 min for adaptation prior to measurement of locomotor activity.

Statistical Analysis

The experimental results are presented as the mean ± standard error of the mean (S.E.M.). Significant differences were determined using one- or two-way ANOVA, followed by the Tukey–Kramer test for multiple group comparisons. All analyses were performed in GraphPad Prism 7 (GraphPad Software, CA, U.S.A.). The criterion for a significant difference was set at p < 0.05.

RESULTS

Receptor Binding Assay and pA₂ Values for NOP Analogs

In the receptor-binding assay, peptide-1, -2, and -3 showed similar IC₅₀ values for the NOP receptor, while peptide-6 showed the highest affinity of all analogs (Table 1). In addition, peptide-6 showed the highest pA₂ value of all peptides in the MVD assay (Table 1).

NOP Analogs Exhibited an Antidepressant-Like Effect in the Tail Suspension Test

To assess whether NOP analogs exhibit antidepressant-like effects in mice, we measured the change in immobility duration using the tail suspension test after administration of the analogs. Administration of peptide-3 and -6 significantly reduced the duration of immobile behavior compared with the vehicle group (p = 0.0279, Fig. 1C; p = 0.0147, Fig. 1F); however, the other analogs showed no significant difference in immobile time compared with the control group (Figs. 1A, 1B, 1D, 1E, 1G, 1H).

Fig. 1. Effects of Nociceptin (NOP) Analogs on Immobility Behavior in the Tail Suspension Test

A–H: Duration of immobility in the tail suspension test 30 min after treatment. The bars represent the mean ± S.E.M. One-way ANOVA: (A) F (3, 50) = 0.8861, p = 0.4548; (B) F (3, 76) = 0.5184, p = 0.6709; (C) F (3, 68) = 4.041, p = 0.0105; (D) F (3, 56) = 1.013, p = 0.3937 (E) (3, 68) = 2.348, p = 0.0803; (F) F (3, 58) = 3.525, p = 0.0204; (G) F (2, 45) = 2.641, p = 0.0823; (H) F (3, 56) = 2.007, p = 0.1234. *p < 0.05, vehicle-treated group (n = 6–24 mice per group).

Peptide-6 Did Not Affect Spontaneous Locomotor Activity in Mice

To exclude the possibility that peptide-6 administration alters locomotion, which in turn could affect the duration of immobility behavior in the tail suspension test, we measured locomotor activity in mice using SUPERMEX. There were no changes in locomotor activity in mice treated with peptide-6 compared with mice in the vehicle group (Fig. 2).

Fig. 2. Effects of Peptide-6 on Locomotor Activity in Mice

Mice were injected with vehicle or peptide-6, and 30 min later, motor activity was recorded for 10 min (n = 17–18 mice per group). The bars represent the mean ± S.E.M. Student’s t-test: t = 0.1494, df = 33, p = 0.8822.

Inactivation of NOP Receptor Is Involved in Peptide-6-Induced Antidepressant-Like Effect in Mice

Intracerebroventricular administration of the selective NOP receptor antagonist, J-113397 (60 nmoL/mouse), showed antidepressant-like effects in mice (p = 0.0437, Fig. 3A). Moreover, the peptide-6-induced antidepressant-like effect was counteracted by the co-administration of NOP (p = 0.0242, Fig. 3B) but not that of NAL (Fig. 3C).

Fig. 3. Effects of NOP and Naloxone (NAL) on the Antidepressant-Like Effect of Peptide-6

(A) The duration of immobility in the tail suspension test 30 min after administration of J-113397, (B) peptide-6 in combination with NOP (B), (C) or NAL. The bars represent the mean ± S.E.M. One-way ANOVA: (A) F (3, 46) = 3.072, p = 0.0369, Fig. 3A. Two-way ANOVA: peptide × treatment: F (1, 43) = 10.55, p = 0.0023; peptide: F (1, 43) = 3.836, p = 0.0567; treatment: F (1, 43) = 0.6597, p = 0.4212, Fig. 3B; peptide × treatment: F (1, 44) = 0.001895, p = 0.9655; peptide: F (1, 44) = 17.8, p = 0.0001; treatment: F (1, 44) = 0.05235, p = 0.8201, Fig. 3C. *p < 0.05, **p < 0.01 vs. vehicle treated group. ^#p < 0.05 vs. peptide-6 treated group (n = 12–14 mice per group).

DISCUSSION

In this study, we showed that peptide-6 exhibited a higher affinity and antagonistic activity for the NOP receptor than the other peptides, as well as an antidepressant-like effect through inactivating the receptor.

NOP receptor agonists have been reported to exhibit depression-like behavior, whereas NOP receptor antagonists have been reported to exhibit antidepressant-like effects.⁸⁾ Hence, NOP analogs were synthesized to evaluate affinity for the NOP receptor, and changes in immobility time after administration were observed in the tail suspension test. Using the receptor-binding assay, peptide-1, -2, and -3 showed similar affinities for the NOP receptor, while peptide-6 showed the highest affinity of all analogs (Table 1). Peptide-6 had a higher pA₂ value for the NOP receptor than the other peptides, indicating that it possesses the strongest antagonistic action (Table 1). Furthermore, peptide-3 and -6 reduced the duration of immobility in mice during the tail suspension test (Figs. 1C, 1F), while peptide-6 did not affect locomotor activity compared with those in the vehicle-treated group (Fig. 2). From these findings, we suggest that the antidepressant-like effect of peptide-6 is a consequence of its high binding affinity and antagonistic activity for the NOP receptor compared with other peptides.

The stimulation of opioid receptors produces antidepressant-like effects, whereas the inhibition of NOP receptors produces antidepressant-like effects.^9,10,19) The present study showed that the selective NOP receptor antagonist J-113397 had antidepressant-like effects in mice (Fig. 3A), indicating that inhibition of the NOP receptor contributes to antidepressant-like effects. We determined whether the antidepressant-like effects of peptide-6 were due to antagonistic effects at NOP receptors or agonistic effects on typical opioid receptors. Consequently, peptide-6-induced antidepressant-like effects were abolished by the co-administration of NOP (Fig. 3B), but not that of NAL (Fig. 3C), suggesting that the antidepressant-like effect of peptide-6 is related to NOP receptor inhibition.

Despite these novel findings, this study has some limitations that must be noted. The detailed mechanism underlying the antidepressant-like effect of peptide-6 remains unclear. NOP suppresses noradrenergic neuronal activity in the locus coeruleus, resulting in reduced noradrenaline release in the basolateral nucleus of the amygdala.²⁰⁾ NOP receptor antagonists enhance noradrenaline release in the basolateral nucleus of the amygdala, but this effect is inhibited by the local infusion of NOP in this region.²¹⁾ Hence, the detailed mechanism of the antidepressant-like effect of peptide-6 should be investigated in the future, with a focus on the noradrenergic nervous system.

In conclusion, the results of this study suggest that peptide-6 exhibits antidepressant-like effects via NOP receptor inhibition. Owing to this, peptide-6, a NOP receptor antagonist, has potential as a lead compound for the development of antidepressant drugs in the future.

Acknowledgments

This study was supported in part by JSPS KAKENHI (Grant Nos: JP22K06866 and JP24K18367).

Conflict of Interest

The authors declare no conflict of interest.

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