Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands

Abstract

Background and purpose: Nociceptin/orphanin FQ (N/OFQ) receptor (NOP) agonists produce anxiolytic-like effects in rodents while antagonists promote antidepressant-like effects. The aim of this study was to investigate the effect on anxiety and depression of NOP receptor partial agonists such as the peptides [F/G]N/OFQ(1-13)NH2 and UFP-113 and the non-peptide AT-090.

Experimental approach: In vitro AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 were tested for their ability to promote NOP/G-protein and NOP/β-arrestin 2 interaction, using a bioluminescence resonance energy transfer assay. In vivo, they were tested in mice in the elevated plus maze (EPM) and in the forced swim (FST) tests. NOP partial agonists effects were systematically compared to those of full agonists (N/OFQ and Ro 65-6570) and antagonists (UFP-101 and SB-612111).

Key results: In vitro, AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 promoted NOP/G protein interaction, with maximal effects lower than those evoked by N/OFQ and Ro 65-6570. AT-090 behaved as a NOP partial agonist also in inducing β-arrestin 2 recruitment, while UFP-113 and [F/G]N/OFQ(1-13)NH2 were inactive in this assay. In vivo, AT-090 induced anxiolytic-like effects in the EPM but was inactive in the FST. Opposite results were obtained with UFP-113 and [F/G]N/OFQ(1-13)NH2.

Conclusions and implications: NOP ligands producing similar effects on NOP/G protein interaction (partial agonism) but showing different effects on β-arrestin 2 recruitment (partial agonism vs antagonism) elicited different actions on anxiety and mood. These results suggest that the action of a NOP ligand on emotional states is better predicted based on its β-arrestin 2 rather than G-protein efficacy.

Keywords: Anxiety; BRET; Depression; Elevated plus maze; Forced swim test; G protein; Mouse; N/OFQ; N/OFQ (PubChem CID: 16131448); NOP receptor partial agonist; SB-612111 (PubChem CID: 10047612); UFP-101 (PubChem CID: 25081457); β-arrestin.

Fig. 1

BRET assay. Effects of N/OFQ (A), Ro 65–6570 (B), UFP-101 (C), AT-090 (D), [F/G]N/OFQ(1–13)NH₂ (E), and UFP-113 (F) on NOP/G-protein and NOP/β-arrestin 2 interaction. Effects are expressed as E/E_max, where E is the effect evoked by the compound and E_max is the maximal effect evoked by N/OFQ. E_max corresponds to a stimulation of 0.33 ± 0.02 BRET ratio units over the baseline for the NOP/G-protein assay and of 0.11 ± 0.01 BRET ratio units over the baseline for the NOP/β–arrestin 2 assay. Data are shown as mean ± sem of 4 separate experiments performed in duplicate.

Fig. 2

Effects of standard compounds in the EPM test. Panels A and B: effect of diazepam (1 mg/kg, i.p., 30 min prior the test) and Ro 65–6570 (0.01–0.1 mg/kg, i.p., 30 min prior the test) in CD-1 mice subjected to the EPM test. Data are mean ± s.e.m. of 12–16 mice per group. Kruskal–Wallis H test followed by the Dunn's post hoc test revealed an effect of treatment both for the percentage of time spent in the open arms (H₍₄₎ = 42.41, A) and the percentage of entries in the open arms (H₍₄₎ = 37.42, B). *p < 0.05 vs vehicle. Panels C and D: effect of SB-612111 (10 mg/kg, i.p., 30 min pre-treatment) per se and against Ro 65–6570 (0.1 mg/kg, i.p., 30 min before starting the test) in CD-1 mice subjected to the EPM test. Data are mean ± s.e.m. of 10–12 mice per group. Two-way ANOVA followed by the Bonferroni's post hoc test revealed an effect of Ro 65–6570 and of the interaction Ro 65–6570 × SB-612111 both for the percentage of time spent in the open arms (F_(1,38) = 5.18 for Ro 65–6570 and F_(1,38) = 4.85 for the interaction, C) and for the percentage of entries in the open arms (F_(1,38) = 5.39 for Ro 65–6570 and F_(1,38) = 4.82 for the interaction, D). *p < 0.05 vs vehicle, #p < 0.05 vs Ro 65–6570. Panels E and F: effect of diazepam (1 mg/kg, i.p., 30 min before starting the test) and Ro 65–6570 (0.1 mg/kg, i.p., 30 min before starting the test) in NOP(+/+) and NOP(−/−) mice subjected to the EPM test. Data are mean ± s.e.m. of 8–10 mice per group. Two-way ANOVA followed by the Bonferroni's post hoc test revealed an effect of treatment and of the interaction treatment × genotype for the percentage of time spent in the open arms (F_(2,47) = 17.68 for treatment and F_(2,47) = 3.86 for the interaction, D) and an effect of treatment for the percentage of entries in the open arms (F_(2,47) = 11.79, E). *p < 0.05 vs vehicle, #p < 0.05 vs NOP(+/+).

Fig. 3

Effects of AT-090 in the EPM test. Panels A and B: dose–response curve to AT-090 (0.001–0.03 mg/kg, i.p., 30 min prior the test) in CD-1 mice subjected to the EPM test. Data are mean ± s.e.m. of 15–17 mice per group Kruskal–Wallis H test followed by the Dunn's post hoc test revealed an effect of treatment both for the percentage of time spent in the open arms (H₍₄₎ = 19.39, A) and the percentage of entries in the open arms (H₍₄₎ = 14.44, B). *p < 0.05 vs vehicle. Panels C and D: effect of AT-090 (0.01 mg/kg, i.p., 30 min prior the test) in NOP(+/+) and NOP(−/−) mice subjected to the EPM test. Data are mean ± s.e.m. of 10 mice per group. Two-way ANOVA followed by the Bonferroni's post hoc test revealed an effect of treatment and of the interaction treatment × genotype for the percentage of time spent in the open arms (F_(1,38) = 10.31 for treatment and F_(1,38) = 6.03 for the interaction, C) and for the percentage of entries. *p < 0.05 vs vehicle, #p < 0.05 vs NOP(+/+).

Fig. 4

Effects of standard compounds and partial agonists of NOP receptor (AT-090, UFP-113, and [F/G]N/OFQ(1–13)NH₂) in the FST. Panel A: effects of nortriptyline (20 mg/kg, i.p., 30 min prior the test), SB-612111 (10 mg/kg, i.p., 30 min prior the test), and Ro 65–6570 (0.1 mg/kg, i.p., 30 min prior the test) on the immobility time of CD-1 mice subjected to the FST. Data are mean ± s.e.m. of 11–16 mice per group. One-way ANOVA followed by the Dunnett's post hoc test revealed an effect of treatment (F_(3,55) = 45.38). *p < 0.05 vs vehicle. Panel B: dose–response curve to AT-090 (0.01–0.1 mg/kg, i.p., 30 min prior the test) on the immobility time of CD-1 mice subjected to the FST. Data are mean ± s.e.m. of 8–12 mice per group. Panel C: effect of UFP-101 (10 nmol, i.c.v., 5 min prior the test) on the immobility time of Swiss mice subjected to the FST. Data are mean ± s.e.m. of 16–17 mice per group. Unpaired Student t test revealed an effect of treatment (t₍₃₁₎ = 3.30). *p < 0.05 vs saline. Panel D: dose–response curve to UFP-113 (0.01–0.1 nmol, i.c.v., 5 min prior the test) in Swiss mice subjected to the FST. Data are mean ± s.e.m. of 10–13 mice per group. One-way ANOVA followed by the Dunnett's post hoc test revealed an effect of treatment (F_(3,47) = 7.90). Panel E: dose–response curve to [F/G]N/OFQ(1–13)NH₂ (0.1–1 nmol, i.c.v., 5 min prior the test) in Swiss mice subjected to the FST. Data are mean ± s.e.m. of 13 mice per group. One-way ANOVA followed by the Dunnett's post hoc test revealed effect of treatment (F_(3,48) = 6.50). *p < 0.05 vs saline.