Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice

Abstract

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Fig. 1.

Basal tail-flick but not hot-plate response latencies are prolonged in βarr2-KO mice. A, Tail flick. Mice of each genotype were assessed for their responsiveness in the warm water tail-flick assay. Response latencies were assessed at 43, 48, and 54°C water temperatures; *p < 0.05, **p < 0.01, and ***p < 0.001 versus WT; ^#p < 0.05 versus βarr2+/−; one-way ANOVA followed by Bonferroni's multiple comparison test; n = 13–20 WT;n = 6–8 βarr2+/−; n = 13–21 βarr2-KO mice. B, Hot-plate. WT and βarr2-KO mice were assessed for paw-withdrawal latencies on the 50, 53, and 56°C hot plate. There were no differences in responses between the littermates (n = 12–27).

Fig. 2.

Enhanced μOR coupling to G-proteins in spinal cord membranes from βarr2-KO mice. Data were analyzed by nonlinear regression using GraphPad Prism software and are presented as the mean ± SEM of four experiments performed in triplicate, wherein WT and βarr2-KO spinal cord membranes were assayed simultaneously. The percentage of agonist-stimulated binding over basal is expressed. In the absence of agonist stimulation, basal [³⁵S]GTPγS binding was as follows: WT, 1835 ± 251 cpm; βarr2-KO, 1675 ± 223 cpm. The curves are significantly different (p < 0.001) compared with two-way ANOVA. DAMGO,d-Ala²-N-Me-Phe⁴-Glycol⁵]enkephalin.

Fig. 3.

Antagonist reversal of enhanced basal tail-flick latencies. Antagonists were injected 20 min before the tail-flick test was performed. Water temperature was 48°C.A, Naltrexone (2 mg/kg, i.p.) antagonizes tail-flick latencies of βarr2-KO and βarr2+/− to that experienced by WT mice.B, Naltrindole (2.5 mg/kg, s.c.) and nor-BNI (5 mg/kg, s.c.) did not affect the enhanced latencies seen in the βarr2-KO mice; *p < 0.01 and **p < 0.001 versus WT basal; ^#p < 0.01 and^§p < 0.05 versus βarr2+/− basal; and ^##p < 0.001 versus βarr2-KO basal; one-way ANOVA followed by Bonferroni's multiple comparison test; n = 6–8 mice per genotype.

Fig. 4.

Morphine induces enhanced and prolonged tail-flick latencies in βarr2-KO mice. Morphine (10 mg/kg, s.c.) was injected, and tail-withdrawal latencies were recorded 15, 30, 45, 60, 90, and 120 min later (54°C water). The genotypes are significantly different (p < 0.0001) by two-way ANOVA;n = 7 mice per genotype.

Fig. 5.

βarr2-KO mice do not develop acute morphine tolerance. Mice were pretreated with either saline or morphine (100 mg/kg, s.c.), and 24 hr later, when they had returned to their basal latencies, mice were administered morphine (10 mg/kg, s.c.). Tail-withdrawal latencies (54°C water) were assessed 30 min after this second injection. Although the WT mice experience tolerance 24 hr after receiving the first injection of morphine (p < 0.001 compared with saline pretreatment), the βarr2-KO mice do not. The βarr2-KO mice experience greater antinociception after morphine than WT mice regardless of whether they were pretreated with morphine or saline (p < 0.001). Analysis is by one-way ANOVA followed by Bonferroni's multiple comparison test;n = 6 mice per genotype.

Fig. 6.

Chronic morphine tolerance in WT and βarr2-KO mice. A, Mice received morphine (10 mg/kg, s.c.) each day; 30 min later, tail-flick latencies were recorded (54°C water). βarr2-KO experience significantly enhanced morphine antinociception compared with WT mice for the first 4 d (*p < 0.001; ^#p < 0.005), with a significant decrease on days 4 and 5 (p < 0.02). Student's t test; n = 7 mice per genotype. Inset, On day 7, mice were placed on the hot plate (56°C), and paw-withdrawal latencies were recorded.B, A morphine dose–response curve was generated in animals on day 1 and again after 7 d of daily morphine treatments. In these studies, mice were not tested daily for antinociceptive latencies. Some points at the lower doses are the averaged responses after cumulative dosing of morphine as described previously (Bohn et al., 2000a). These points were similar to results obtained with an acute dose of the drug and were therefore averaged together. ED₅₀ values were calculated via nonlinear regression analysis (GraphPad Prism), and 95% confidence intervals are as follows: day 1 WT, 10.3 (7.6–13.7); βarr2-KO, 4.6 (4.5–4.8); day 7 WT, 75.8 (51.3–112.1); βarr2-KO, 34.5 (33.8–35.3) mg/kg.

Fig. 7.

Chelerythrine reverses morphine tolerance in βarr2-KO mice. A, Mice that had been treated for 7 d with morphine were then given one of the following combinations: vehicle for 10 min plus morphine (10 mg/kg, s.c.) for 30 min, chelerythrine (5 mg/kg, i.p.) for 10 min plus vehicle for 30 min, or chelerythrine (5 mg/kg, i.p.) for 10 min plus morphine (10 mg/kg, s.c.) for 30 min. At the end of the 40 min total treatment time, antinociceptive latencies were tested (54°C water). **p < 0.001 versus all points; one-way ANOVA followed by Bonferroni's multiples comparison test;n = 6 (Morphine);n = 3 (Chelerythrine);n = 9 (Chelerythrine + Morphine).B, Naive mice were treated with either vehicle for 10 min plus morphine (5 mg/kg, s.c.) for 30 min or chelerythrine (5 mg/kg, i.p.) for 10 min plus morphine (5 mg/kg, s.c.) for 30 min before antinociceptive testing. **p < 0.001 versus WT;n = 5–6, as assessed in A.