Sex- and β-arrestin-dependent effects of kappa opioid receptor-mediated ethanol consumption

Abstract

The kappa opioid receptor is a known regulator of ethanol consumption, but the molecular mechanisms behind its actions have been underexplored. The scaffolding protein β-arrestin 2 has previously been implicated in driving ethanol consumption at the related delta opioid receptor and has also been suggested to be a driver behind other negative kappa opioid receptor mediated effects. Here, we used kappa opioid agonists with different efficacies for recruiting β-arrestin 2 and knockout animals to determine whether there is a role for β-arrestin 2 in the modulation of voluntary ethanol consumption by the kappa opioid receptor. We find that an agonist with low β-arrestin 2 efficacy more consistently lowers ethanol consumption than agonists with high efficacy for β-arrestin 2. However, knockdown of β-arrestin 2 amplifies the ethanol consumption-promoting effects of the arrestin-recruiting kappa agonists U50,488 and nalfurafine. We control for potentially confounding sedative effects at the kappa opioid receptor and find that β-arrestin 2 is not necessary for kappa opioid receptor-mediated sedation, and that sedation does not correlate with effects on ethanol consumption. Overall, the results suggest a complex relationship between agonist profile, sex, and kappa opioid receptor modulation of ethanol consumption, with little role for kappa opioid receptor-mediated sedation.

Keywords: Biased signaling; HS666; Intermittent access; Locomotor; Nalfurafine; Two-bottle choice.

Fig. 1.. HS666, nalfurafine and U50,488 have distinct in vitro pharmacologies, particularly in relation to βarr2 recruitment.

A) cAMP Glosensor assay showing κOR-mediated inhibition of forskolin-stimulated cAMP production in HEK cells. B) DiscoverX PathHunter assay showing agonist-triggered recruitment of βarr2 to the κOR in U2OS cells. See Table 1 for fit values.

Fig. 2.. κOR agonists with distinct βarr2-recruitment efficacies display divergent modulation of ethanol intake in a two-bottle choice voluntary ethanol consumption test in WT C57BL/6 mice.

A-F) Weekly baseline and treatment day ethanol consumption for males (A-C) and females (D-F) in response to doses of U50,488 (A, D), HS666 (B, E), and nalfurafine (C, F). G-I) Area under the curve (A.U.C.) analysis of A-F to evaluate sex differences in cumulative response to drugs relative to vehicle. Plots are mean ± SEM. A-G) **, p < 0.01; ***, p < 0.001 with respect to baseline. G-I) * p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 10⁻⁴; n = 8-11.

Fig. 3.. Loss of βarr2 tends to elevate ethanol consumption in response to agonism by U50,488 or nalfurafine in a two-bottle choice voluntary ethanol consumption paradigm.

A-D) Weekly baseline and treatment day ethanol consumption between for βarr2 KO males (A, B) and βarr2 KO females (C, D) in response to doses of U50,488 (A, C) and nalfurafine (B, D). E-F) Area under the curve (A.U.C.) analysis for (A-D) comparing net effects of U50,488 and nalfurafine relative to vehicle in βarr2 KO mice to WT data in Fig. 2. Plots are mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for indicated comparison; n = 10-12.

Fig. 4.. Nalfurafine treatment tends to elevate ethanol consumption in the intermittent access two-bottle choice paradigm in WT and βarr2 KO male mice.

A-D) Ethanol consumption on treatment day and during weekly baseline for WT males (A), and βarr2 KO males (B), WT females (C), and βarr2 KO females (D) in response to doses of nalfurafine. Results shown are for the first four hours of the 24-hour drinking sessions, corresponding to the 4 hours of ethanol access in the two-bottle choice paradigm in Figures 2 and 3. E) Area under the curve (A.U.C.) analysis for (A-D) comparing net effect of nalfurafine relative to vehicle. When response to vehicle is accounted for, the net effect of nalfurafine in this model is small. *, p < 0.05; **, p < 0.01 with respect to weekly baseline.

Fig. 5.. κOR agonists induce hypolocomotion independently of their ability to recruit βarr2.

A-B) κOR-mediated hypolocomotion in male and female WT and βarr2 KO mice by U50 (A) and nalfurafine (B), normalized to vehicle. C) HS666-mediated hypolocomotion in male and female WT mice, normalized to vehicle. Symbols above graphs show results of statistical tests on unnormalized data summarized in Table S3 and denote differences from vehicle. Plots are mean ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n = 5-10.