Involvement of GRK2 in modulating nalfurafine-induced reduction of excessive alcohol drinking in mice

Abstract

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine’s efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.

INTRODUCTION

In humans, neurobiological studies have provided strong supportive results, demonstrating that among multiple actions of alcohol, chronic alcohol exposure altered mu-opioid receptor [MOR] and kappa-opioid receptor [KOR] activities in the CNS [1] [2]. In rodents, both the MOR/endorphin and KOR/dynorphin systems are profoundly activated after long-term alcohol consumption, which are involved in the positive and negative reinforcing aspects of alcohol intake, respectively [2] [3]. Alcohol exposure increased G protein-coupled receptor kinase 2 [GRK2] expression and function in hippocampus and midbrain, leading to an increased association of MOR with GRK2, a GRK2 upregulation-induced MOR desensitization and an altered morphine reward [4] [5] [6]. Pharmacologically, intra-cerebral injection of GRK2 inhibitor altered morphine rewarding effect in alcohol-experienced mice, further indicating potential interactions among alcohol, GRK2 activation and MOR [6]. GRK2 has also been suggested to involve in cocaine-induced behaviors [7] [8].

However, it has not been studied if the GRK family is altered after chronic alcohol intake in the nucleus accumbens shell (NAcs), a critical brain region highly involved in rewarding regulations by both MOR and KOR activity. In the present study, therefore, we analysed gene transcripts using an unbiased RNA sequencing (RNA-seq) approach in the nuclear compartment (more sensitive to the gene transcriptional changes than the cytoplasmic compartment) [9] [10] and observed a significant up-regulation of nuclear grk2 [adrbk1] transcript levels in the NAcs of mice after chronic excessive alcohol drinking (about 10–25 g/kg/day).

Of interest, a recent study has found that typical KOR agonist U50,488H-induced analgesia is enhanced by selective GRK2/3 inhibitor CMPD101 in female mice [11], suggesting that GRK2 exhibits an inhibitory effect on the G protein-mediated signaling required for KOR activity on analgesic effects. In cultured cardiomyocytes and mouse heart, the U50,488H-induced KOR activity was desensitized by overexpression of GRK2, further suggesting a potential interaction between GRK2 and KOR [12]. Nalfurafine, a KOR full agonist, produced analgesic effects and decreases excessive alcohol drinking through G-protein-mediated KOR signaling, with a lack of side effects [13] [14] [15] [16]. Based on these, we proposed a novel hypothesis that the NAcs grk2 increase could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Furthermore, whether nalfurafine, a KOR agonist clinically utilized as an anti-pruritus drug, can be redesigned to have clinical efficacy on alcoholism treatment will be helped by a better understanding of its distinct signaling pathways. Therefore, the present study was designed to identify potential intracellular signaling mechanisms responsible for the effect of nalfurafine on alcohol intake with a goal to provide potential insights to guide the better development. Accordingly, we evaluated the functional effect of selective GRK2/3 inhibitor CMPD101 on alcohol drinking and investigated whether pharmacological blockade of GRK2/3 could alter the nalfurafine-induced reduction of alcohol drinking. We found that though CMPD101 alone had no effect on alcohol consumption, pretreatment with the GRK2/3 inhibitor enhanced the reduction in excessive alcohol intake induced by nalfurafine. Together, our results provide initial and supportive evidence relevant to the transcriptional change of the grk2 gene expression in the NAcs of mice after excessive alcohol drinking, and identify an alcohol-induced GRK2 signaling mechanism, leading to an altered response to the G protein-biased KOR agonist nalfurafine.

MATERIAL AND METHODS

ANIMALS.

Male adult C57BL/6J (B6) mice (8 weeks of age) were placed on a 12-hour reverse light-dark cycle (lights off at 7:00 am) and housed individually in ventilated cages with steel lids and filter tops and given ad libitum access to food and water.

MATERIALS.

15% ethanol solution (v ⁄ v) was prepared from 190-proof absolute ethyl alcohol (Pharmco-AAPER, Brookfield, CT, USA) and dissolved in tap water. Nalfurafine (NIDA Division of Drug Supply and Analytical Services) was dissolved in saline for systemic i.p. administration. CMPD101 (Sigma-Aldrich) was dissolved in 5% DMSO, 5% Cremaphor EL and 90% saline for i.p. administration.

PROCEDURES.

The chronic (3 weeks) intermittent access (CIA) drinking procedure (Table S1).

In this two-bottle free choice paradigm, mice had access to alcohol for 24 hours every other day for 3 weeks. The basic paradigm with our modifications was as reported before [15]: briefly, both the 15% alcohol solution and water tubes were provided starting at 3 hours after lights off. After 4, 8 and 24 hours of alcohol access, both the water and alcohol intakes were recorded. Following the 3-week CIA procedure the mice had high alcohol intake and preference.

Nuclear RNA Extraction.

In the experiment of RNA-seq analysis (Table S1A), the mice were randomly assigned to the water control and the alcohol groups (n=12/each). Mice were sacrificed 1 day after the last CIA session; the NAcs was immediately dissected. The snap-frozen NAcs pooled from two mice were fractionated into cytoplasmic and nuclear phases [10]. The nuclear pellet was treated with RNase-free DNase-1, followed by proteinase K. Finally, the total nuclear RNA was isolated using the miRNeasy kit (Qiagen), and the quality and quantity of nuclear RNA from each sample was determined using an Agilent 2100 Bioanalyzer. This method permits efficient lysis of the brain tissues as evidenced by absence of cytoplasmic tRNA in the nuclear extract fraction, with minimal rupture of nuclei as indicated by absence of DNA in cytoplasmic extract fraction, even with very vigorous homogenization.

RNA-seq library preparation and sequencing.

Starting with 100 ng of nuclear RNA samples, both the library preparation and sequencing was performed by Genomic Resource Center at Rockefeller University: (1) Illumina’s TruSeq® Stranded Total RNA Library Prep Kit with Ribo-Zero following manufacturer protocol: the libraries were validated with Agilent Tape Station High Sensitivity DNA kits and normalized; and (2) Illumina NextSeq 500 sequencer using high output V2 reagents and NextSeq Control Software v1.4 generates 75 bp paired end reads, following manufacture protocol. As each lane of NextSeq provides 400 million reads, the 12 samples on one NextSeq lane were set up with 30 million+ reads per sample.

RNA-seq data quality assessment and differential transcript analysis.

The fastq files were generated by configuring BclToFastq.pl from CASAVA v1.8.2, and then examined using FASTQC. The reads were aligned to the mouse reference genome (version mm10) using STAR v2.3 aligner with default parameters. The alignment results were evaluated through qualimap v2.2 [https://academic.oup.com/bioinformatics/article/32/2/292/1744356]. Aligned reads were summarized through feature Counts with the gene model from Ensembl at gene level. Only the protein coding genes were used for the analysis of this study. Principal Component Analysis (PCA) was then applied to the normalized count of all the samples from the NAcs to detect potential outliers. Ingenuity Pathway Analysis (IPA), an advanced bioinformatics tool, was also used to obtain the potential interaction of alcohol with the GRK pathway. We used DESeq2, a method for differential analysis of RNA-seq data to estimate fold-change and significance testing. For significance testing, DESeq2 uses a Wald test.

Alcohol drinking with administration of CMPD101, nalfurafine or CMPD101+nalfurafine after CIA (Table S1B).

The first objective was to determine dose-dependent effects of GRK2/3 inhibitor CMPD101 on alcohol drinking. The range of CMPD101 doses (0, 0.1 or 0.3 mg/kg, i.p.) was based on our pilot study. On the test day (1-day after CIA), alcohol was presented 15 min after CMPD101 or vehicle injection (n=6–8), and then alcohol and water intake values were recorded. The second objective was to determine dose-related effects of nalfurafine on alcohol drinking. On the test day, nalfurafine (0, 1, 3 or 10 μg/kg, i.p.) or vehicle injection was administered 5 min before a drinking session (n=6–8). The range of nalfurafine doses and time point were based on our previous publication [15]. Finally, we tested whether CMPD101 pretreatment could alter the nalfurafine-induced decrease in alcohol drinking, to examine whether the nalfurafine effects were modulated by CMPD101. The CIA mice were pretreated with CMPD101 (0 or 0.3 mg/kg, i.p.) 15 min before the drinking session, followed by nalfurafine (0, 1, 3 or 10 μg/kg, i.p.) 5 min before a drinking session (n=7–8).

Data analysis.

Power analyses were performed to determine the number of mice (about 6–8 mice per group) required to provide statistically significant RNA-seq and behavioral results, based on the levels of differences reported before (see details in Supplementary Information section) [15]. For RNA-seq experiment, an adjusted p-values of less than 0.05 (FDR<0.05) was used to select genes that have a significant expression change. For dose-response analysis on CMPD101, nalfurafine or CMPD101+ nalfurafine, group differences were analyzed using 2-way ANOVA for pretreatment (vehicle or CMPD101) and treatment with different doses of nalfurafine (1, 3 or 10 μg/kg). In the experiments with nalfurafine after CMPD101 pretreatment, group differences were analyzed using 2-way ANOVA for pretreatment (vehicle vs CMPD101) and for treatment (vehicle vs nalfurafine). All the ANOVAs were followed by Newman-Keuls post-hoc tests, and accepted level of significance was p<0.05.

RESULTS

Alcohol intake and preference.

After 3 weeks of chronic alcohol drinking, the mice had alcohol intake averaging ~15 g/kg/day, with high preference ratio (~0.8). The detailed data are presented in Table S2.

Effects on nuclear transcript levels in the NAcs after CIA.

A. GRK family genes.

RNA-seq analysis revealed a significant change in response to CIA (Table 1): grk2 gene showed a significant up-regulation in the nuclear transcript levels, with no significant change of other grk genes. To further analyze the relationship between the last 24-hour alcohol intake in session 10 and subsequent individual changes in the nuclear transcript levels, grk2 gene was examined to determine whether there was any correlation of the altered nuclear transcript levels in relationship with the individual amount of alcohol intake. This analysis revealed no significant correlation (Figure S1).

Table 1.

Nuclear transcript levels of G protein-coupled receptor kinase (grk) family genes in the nucleus accumbens shell were altered after alcohol-free day from 3-week chronic intermittent access (CIA) excessive alcohol drinking (FDR < 0.05). FC, fold change; FDR, false discovery rate; average DESeq2 normalized count (NC) for water and alcohol -exposed mice (n=6 samples for each group).

Gene	Fold	p-Value	FDR	Water NC	Alcohol NC	Name
grk2 [adrbk1]	1.17	0.002	0.041	1115	1322	G Protein-Coupled Receptor Kinase 2
grk3 [adrbk2]	1.12	0.047	0.188	1014	1155	G Protein-Coupled Receptor Kinase 3
grk4	1.08	0.319	0.546	49	53	G protein-Coupled Receptor Kinase 4
grk5	1.12	0.004	0.051	269	303	G protein-Coupled Receptor Kinase 5
grk6	1.08	0.060	0.215	581	630	G protein-Coupled Receptor Kinase 6

B. RAS-related protein family genes.

rab8b and rab40c genes showed significant up-regulations in the nuclear transcript levels, with no change of others (Table S3).

C. G protein subunits family genes.

Only gnal and gnao1 genes showed significant up-regulations in the nuclear transcript levels (Table S4).

D. Arrestin beta (arrb), serine/threonine-protein kinase (akt), protein kinase C (prkca), dynamin (dnm) and protein phosphatase 2 catalytic subunit genes.

As shown in Table S5, there was no change after CIA.

No effects of CMPD101 alone on alcohol intake or preference.

At 0.1 or 0.3 mg/kg, there was no significant effect of CMPD101 on either intake or preference at 0–4 hours after alcohol access (n = 6–8) (control mice: 5.1 ± 0.21 g/kg; CMPD101 at 0.1 mg/kg: 5.2 ± 0.40 g/kg; and CMPD101 at 0.3 mg/kg: 4.9 ± 0.38 g/kg) or preference ratio (control: 0.81 ± 0.05; CMPD101 at 0.1 mg/kg: 0.82 ± 0.04; CMPD101 at 0.3 mg/kg: 0.79 ± 0.03). At the 5–8 or 9–24 hour intervals, there were no significant effects on alcohol dirking (Table S6).

Administration of nalfurafine decreased alcohol consumption after CIA.

At 1, 3 and 10 μg/kg, nalfurafine decreased alcohol intake at 0–4 hours in a dose-related manner (Figure 1A, left) (as percentages of baseline alcohol intake): 2-way ANOVA showed a significant effect of nalfurafine on alcohol intake [F(1,47)=34, p<0.001] and Newman-Keuls post hoc analysis showed that the mice treated with nalfurafine at 10 μg/kg had significantly less alcohol intake than the vehicle control [p<0.05]. For preference ratio, there was also a significant decease after nalfurafine [2-way ANOVA, F(1,47)=56, p<0.001] at 10 μg/kg [p<0.05] (Figure 1B, left). At the 5–8 or 9–24 hour intervals, there were no significant effects of nalfurafine (Table S7).

Figure 1.

Dose responses of nalfurafine (0, 1, 3 or 10 μg/kg) alone or with pretreatment with GRK2/3 inhibitor CMPD101 (0 or 0.3 mg/kg) on reducing alcohol intake (A) and alcohol preference ratio (B) in mice after 3-week chronic excessive drinking. Data were collected at the 0–4 hour interval on the baseline and testing day and are expressed as a percentage of baseline alcohol intake to account for the differences in baseline that contribute to variation between experiments. * p<0.05 or ** p<0.01 vs. vehicle control (0 μg/kg NFF + 0 mg/kg CM); #p<0.05 between 10 μg/kg NFF treatment groups (n=6–8).

CMPD101 pretreatment enhanced the nalfurafine-induced reduction in alcohol intake.

In a pilot study, CMPD101 at 0.1 mg/kg had no effect on the nalfurafine (1, 3 or 10 μg/kg)-induced decrease of alcohol drinking (Table S8).

Figure 1(right) presents a dose response (as percentages of baseline alcohol intake) of nalfurafine (0, 1, 3 or 10 μg/kg) pretreated with CMPD101 (0 or 0.3 mg/kg) in alcohol intake and preference at 0–4 hours. For alcohol intake (Figure 1A, right), Newman-Keuls post hoc analysis revealed that (1) compared with the control group (CMPD101 0 mg/kg + nalfurafine 0 μg/kg), after pretreatment with CMPD101 (0.3 mg/kg), the mice treated with nalfurafine at 3 and 10 μg/kg had significant less intakes [2-way ANOVA followed by Newman-Keuls post hoc analysis: p<0.05 and p<0.01, respectively]; and (2) compared with the group treated with nalfurafine at 10 μg/kg alone, pretreatment with CMPD101 at 0.3 mg/kg enhanced the effect of nalfurafine at 10 μg/kg dose [p<0.05]. For preference ratio (Figure 1B, right), 2-way ANOVA followed by Newman-Keuls post hoc analysis revealed that compared with the control group, the mice treated with CMPD101 followed by nalfurafine at 3 and 10 μg/kg had significant less preference ratios [p<0.05 and p<0.01, respectively].

Figure 2 shows the originally recorded alcohol and water intakes with calculated preference ratio at 0–4 hours in the experiment of nalfurafine at 10μg/kg alone or with CMPD101 pretreatment at 3 mg/kg. For alcohol intake (Figure 2A), 2-way ANOVA showed significant effects of nalfurafine [F(1,27)=16, p<0.001] and CMPD101 pretreatment [F(1,27)=4.8, p<0.05], with a significant interaction between the CMPD101 pretreatment and nalfurafine [F(1,27)=11, p<0.005]. Newman-Keuls post-hoc tests showed that nalfurafine alone or with CMPD101 pretreatment significantly decreased alcohol intakes [Vehicle vs. Nalfurafine, p<0.05; Vehicle vs. CMPD101+Nalfurafine, p<0.01], and the CMPD101 pretreatment enhanced the effect of nalfurafine [CMPD101+Nalfurafine vs. Nalfurafine, p<0.05]. For water intake (Figure 2B), 2-way ANOVA showed a significant effect of nalfurafine [F(1,27)=12, p<0.005], and the mice treated with CMPD101+Nalfurafine had more water intake than the ones with vehicle [p<0.01] or nalfurafine alone [p<0.05]. For the preference ratio (Figure 2C), there was a significant effect of nalfurafine [2-way ANOVA, F(1,27)=21, p<0.001], and the mice treated with nalfurafine alone or with CMPD101 pretreatment had less preference ratios than the vehicle control [p<0.05 and p<0.01, respectively]. At the 5–8 or 9–24 hour intervals, there were no significant effects on alcohol dirking (Table S7).

Figure 2.

Effects of nalfurafine alone or with pretreatment with GRK2/3 inhibitor CMPD101 on alcohol intake (A), water intake (B), and preference ratio (C) in mice after alcohol-free day from chronic (3-week) excessive drinking (n=7–8). (1) Vehicle (Veh) group: mice received two vehicle injections (i.p.) before the drinking session; (2) Nalfurafine (NFF) group: mice received one nalfurafine injection (10 μg/kg, i.p.) 5 min before the drinking session; (3) CMPD101 (CM) group: mice received one CMPD101 injection (0.3 mg/kg, i.p.) 15 min before the drinking session; and (4) CMPD101+Nalfurafine (CM+NFF) group: mice pretreated with CMPD101 (0.3 mg/kg, i.p.) 15 min before the drinking test, followed by one nalfurafine (10 μg/kg) injection 5 min before the drinking test. Alcohol (15%) and water intake values were recorded after 4 hours of alcohol access. *p<0.05 or ** p<0.01 vs. vehicle group; +p<0.05 between NFF and CM+NFF treatment groups.

DISCUSSION

In the present study, we found a significant increase in grk2 (but not other grks) gene transcript levels in the NAcs of mice after chronic excessive alcohol drinking (Table 1). Our result that alcohol selectively altered the NAcs grk2 is in line with previous studies showing the alcohol-induced GRK2 increases at protein phosphorylation levels in other brain regions [5] [6]. Activation of MOR by beta-endorphin [encoded by Pomc] produces rewarding effects, and beta-endorphin, like MOR agonists, induces the desensitization of MOR through GRK2-mediated mechanism [4]. Given the fact that beta-endorphin release and Pomc expression in the NAcs are increased by alcohol [17] [18], our finding suggests that alcohol may initially activate GRK2 by beta-endorphin, which may explain that KOR responsivity and function may be consequently changed by an up-regulation of GRK2. When examining the effect of pharmacological blockade of GRK2/3, we found that selective inhibitor CMPD101 alone did not affect alcohol drinking, which is consistent with previous studies showing that GRK2/3 alone does not play a tonic role in dopamine release, analgesia or rewarding regulation induced by MOR or KOR activation [6] [11].

GRK2 interacting with MOR or KOR increases arrestin-dependent signaling, with decreasing their G protein-mediated signaling [11] [19]. Indeed, Abraham et al demonstrated that KOR/Gβγ-mediated intracellular signaling effects are enhanced following GRK2/3 inhibition, and GRK2 binds Gβγ to decrease KOR-mediated dopamine release inhibition in the striatum of female mice, indicating a direct interaction between GRK2 and KOR [11]. Nalfurafine, a G protein-biased KOR agonist, decreases alcohol consumption probably through interacting with G protein-mediated KOR signaling, with a lack of side-effects [15]. Then, we further tested a new hypothesis that pharmacological inhibition of GRK2 would promote G protein-mediated signaling effects of the G protein-biased KOR agonist nalfurafine, and enhance the nalfurafine-induced reduction in alcohol drinking. Indeed, we found that the nalfurafine effect was increased by selective GRK2/3 inhibitor CMPD101 in a dose-dependent manner (Figure 1). The CMPD101+nalfurafine-induced reduction in alcohol intake (Figure 2A) was coupled with a significant compensatory increase in water intake (Figure 2B) [resulting in no change in total fluid intake], suggesting the absence of a nonspecific sedative effect by the treatment. To our knowledge, this is the first study showing that the function of G protein-biased KOR agonist nalfurafine on decreasing alcohol consumption is under the GRK2/3 inhibition. In support of this concept, previous studies have found a sensitized G protein-mediated KOR function in female mice or in cultured mouse heart after GRK2/3 inhibition [11] [12]. Though our initial and preliminary evidence supported a potential role for the NAcs grk2 involved in the nalfurafine-induced reduction in alcohol drinking, further experiments are necessary to fill important knowledge gaps such as the specific neurons in the NAc shell involved in the KOR and GRK2 interactions. At least, our results of the pharmacological studies provide functional validation on the grk2 transcriptional changes.

We also examined whether chronic excessive alcohol drinking was enough to alter transcriptional activity of other functional protein genes that are related to grk2 [6] [20] and found that RAS-related protein (rab8b and rab40c) and G protein subunit (gnal and gnao1) gene transcripts were increased (Table S3, Table S4). Our nuclear RNA-Seq data may represent nascent transcriptional activity of grk2 and related genes in response to chronic excessive alcohol drinking, enabling the identification of nascent activity-associated gene transcripts.

After chronic alcohol exposure, endogenous KOR/dynorphin activity is increased in several neuronal regions (including the NAc, bed nucleus of the stria terminalis and central amygdala), which produces anxiety- or depression-like behavior, aversion and dysphoria, and enhances alcohol drinking, seeking and “relapse” [21] [22] [23] [24]. Therefore, it is reasonable to speculate that the increased grk2 expression and GRK2 activity in the NAcs of alcohol-experienced mice enhanced arrestin-mediated KOR signaling, which could exaggerate the KOR/dynorphin response triggered by stress into aversion, anhedonia, depression/anxiety-like behavior, with consequent excessive alcohol drinking. Nalfurafine, however, is a potent G protein-biased KOR agonist and can compete with the stress-induced dynorphins to bind KOR, thereby reducing undesired effects induced by dynorphins, perhaps by reducing arrestin-mediated KOR signaling. Taken together, nalfurafine exhibits different molecular, cellular, and behavioral properties than typical KOR agonists [13] [16] [25] [26] and our studies provide support for development of nalfurafine as an anti-addiction medication [15] [27].

Though the nuclear transcripts studied here were highly correlated with gene transcriptional profile, protein changes (assayed by Western blot) are more relevant to protein translational activity and bio-function. Although our pharmacological data with CMPD101 provides initial data relevant to a potential role for the GRK2/3 in nalfurafine effects, and the experiments to determine the effects of CMPD101 on GRK2/3 protein function and grk family gene expression after excessive alcohol drinking could provide further understanding of interactions between GRK2/3, KOR and alcohol drinking behaviors. The above limitations warrant the need for further analyses.

As CMPD101 selectively inhibits both GRK2 and GRK3 activity, with little effect on GRK1 or GRK5 [28], we could not determine if there is a specific involvement of GRK2 or GRK3 for the enhancing effects of CMPD101 on nalfurafine. However, it has been found that GRK3 is not essential in modulation of KOR activation [11]. As global genetic knockout of GRK2 causes death during mouse embryonic development [29], the limit prevents us to study the effect of nalfurafine on alcohol drinking using GRK2 knock-out mice.

Conclusion:

Our characterization of nuclear transcriptional activity represents the further understanding of chronic alcohol exposure in which gene expressions take place in the NAcs: an increase in grk2 (but not grk3) gene transcript, which may desensitize the responses to G-protein biased KOR signaling activated by nalfurafine. The result of a new set point of the grk2 gene transcription activities may also contribute to individual vulnerability to excessive alcohol drinking triggered by stress-induced KOR activation, as typical KOR agonists triggers mTORC1 [25], MAPK [30] and RGS [31] pathways in a region-specific manner, and all of these have been found to be directly or indirectly involved in the KOR induced-aversion. Different from typical KOR agonists, however, activation of KORs by nalfurafine reduces alcohol intake with less side effects, and whether the G protein-biased KOR agonist nalfurafine can be redesigned to have clinical utility will be helped by a better understanding of its distinct signaling mechanisms as studied here.

Highlights:

• Excessive alcohol drinking increased grk2 expression in the NAc shell.

• No effect of GRK2/3 inhibitor CMPD101 alone on alcohol drinking.

• Nalfurafine decreased alcohol consumption.

• CMPD101 enhanced the nalfurafine-induced reduction of drinking