The role of beta-arrestin2 in the severity of antinociceptive tolerance and physical dependence induced by different opioid pain therapeutics

Abstract

Ligands acting at the same receptor can differentially activate distinct signal transduction pathways, which in turn, can have diverse functional consequences. Further, receptors expressed in different tissues may utilize intracellular signaling proteins in response to a ligand differently as well. The mu opioid receptor (MOR), which mediates many of the pharmacological actions of opiate therapeutics, is also subject to differential signaling in response to diverse agonists. To study the effect of diverse agonists on MOR signaling, we examined the effects of chronic opiate treatment on two distinct physiological endpoints, antinociceptive tolerance and physical dependence, in mice lacking the intracellular regulatory molecule, βarrestin2. While βarrestin2 knockout (βarr2-KO) mice do not become tolerant to the antinociceptive effects of chronic morphine in a hot plate test, tolerance develops to the same degree in both wild type and βarr2-KO mice following chronic infusion with methadone, fentanyl, and oxycodone. Studies here also assess the severity of withdrawal signs precipitated by naloxone following chronic infusions at three different doses of each opiate agonist. While there are no differences in withdrawal responses between genotypes at the highest dose of morphine tested (48 mg/kg/day), the βarr2-KO mice display several less severe withdrawal responses when the infusion dose is lowered (12 or 24 mg/kg/day). Chronic infusion of methadone, fentanyl, and oxycodone all lead to equivalent naloxone-precipitated withdrawal responses in both genotypes at all doses tested. These results lend further evidence that distinct agonists can differentially impact on opioid-mediated responses in vivo in a βarrestin2-dependent manner.

Figure 1

Thermal (54°C hot plate) antinociceptive response s in WT and βarr2-KO mice in response to chronic infusion over a 5 day period with (A) morphine (48 mg/kg/day, s.c.; two-way ANOVA with Bonferroni post-hoc analysis for genotype effect compared at at each day: P<0.0001, n=9 WT, 8 KO); (B) methadone (96 mg/kg/day, s.c; n=7 WT, 6 KO); (C) fentanyl (3.2 mg/kg/day, s.c., n=5 WT, 5 KO); or (D) oxycodone (25 mg/kg/day, s.c., n=6 WT, 6 KO). Responses were measured on day 1, 3, and 5 following pump implantation. Data are presented as the mean ± S.E.M. For B-D, two-way ANOVA revealed no differences for genotype effect (P>0.05) and for A-D, one-way ANOVA for day effect reveals differences for WT: *P<0.05, **P<0.01, ***P<0.001; for KO: ^#P<0.05, ^##p<0.01, ^###P<0.001, Bonferroni post-hoc analysis.

Figure 2

Cumulative-dose response curves in WT and βarr2-KO mice in response to chronic (A) morphine (48 mg/kg/day, s.c.; two-way ANOVA analysis for genotype effect compared at day 1: P<0.0001, WT vs KO: *P<0.05, **P<0.01, Bonferroni post-hoc analysis, n=8-11 WT, 8-12 KO), (B) methadone (96 mg/kg/day, s.c., n=6 WT, 6 KO), (C) fentanyl (3.2 mg/kg/day, s.c., n=5 WT, 5 KO), or (D) oxycodone (25 mg/kg/day, s.c., n=5 WT, 5 KO) infusion over a 7 day period. Dose-response curves were determined using a cumulative dosing scheme on day 1 before osmotic pump implantation, and then again after 7 days of chronic drug infusion. Data are presented as the mean ± S.E.M. For B-D, two-way ANOVA analysis revealed no difference for genotype effect (P>0.05).

Figure 3

Naloxone-precipitated (0.5 mg/kg, i.p.) withdrawal jumps and global scores following 7 days of chronic infusion with (A) morphine (12, 24, 48 mg/kg/day, s.c.; two-way ANOVA analysis for genotype effect for jumps (P=0.0180) and global score (P=0.004), WT vs KO: *P<0.05, ***P<0.001, Bonferroni post-hoc analysis, n=8-11 WT, 7-10 KO), (B) methadone (48, 60, 72 mg/kg, s.c., n=8-10 WT, 8-10 KO), (C) fentanyl (0.8, 1.6, 3.2 mg/kg, s.c., n=7-11 WT, 6-10 KO) or, (D) oxycodone (12.5, 25, 75 mg/kg, s.c., n=5-6 WT, 5 KO). Immediately following naloxone administration, withdrawal signs were observed and scored over a 30 min period. Data are presented as the mean ± S.E.M. For methadone, fentanyl, and oxycodone, two-way ANOVA analysis revealed no differences for genotype effect for both jumps (P>0.05) and global score (P>0.05).