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. 2011 Apr 12:7:24.
doi: 10.1186/1744-8069-7-24.

Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

Hoa Lam  1 , Matthew Maga, Amynah Pradhan, Christopher J Evans, Nigel T Maidment, Tim G Hales, Wendy Walwyn
Affiliations

Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

Hoa Lam et al. Mol Pain. .

Abstract

Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs) over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2) augments the constitutive coupling of μ receptors to voltage-activated Ca²+ channels in primary afferent dorsal root ganglion neurons from β-arr2⁻/⁻ mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2⁻/⁻ mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2⁻/⁻ mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2⁻/⁻ and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

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Figures

Figure 1
Figure 1
Increased facilitation in early post-natal and adult DRG neurons. An increase in Ba2+ current facilitation following a depolarizing pre-pulse to 80 mV is consistent with constitutive coupling of GPCRs with voltage-dependent Ca2+ channels (VDCCs) through Gβγ subunits. Facilitation was quantified by a two-step protocol comparing the current amplitude with (P2) or without a pre-pulse (P1), the P2/P1 ratio. A. Early post-natal DRG neurons i. Whole cell patch clamp recordings for all experiments were conducted in small DRGs (highlighted by the arrow, the scale-bar represents 10 μM), the population of cells most likely to express the μ receptor. ii. Exemplar recordings from β-arr2+/+ and β-arr2-/- cells, in which GTP-γ-S was included in the intracellular recording solution, demonstrate the relative increase in current following a depolarizing pre-pulse to 80 mV (P2). Horizontal and vertical scale-bars represent 20 ms and 0.4 nA respectively. iii. The P2/P1 ratio reveals increased facilitation in βarr2-/- but not β-arr2+/+ early post-natal neurons. This difference was enhanced in the presence of GTP-γ-S and absent in neurons lacking both β-arr2 and the μ receptor (μβarr2-/-). B. Adult DRG neurons. i. Similar to the early post-natal DRGs, recordings were made in small DRG neurons from adult mice (highlighted by the arrow, the scale-bar represents 10 μM). ii. Exemplar recordings from adult DRG neurons in which GTP-γ-S was included in the intracellular recording solution, similarly demonstrate an increased current after the depolarizing pre-pulse (P2) in βarr2-/- neurons. Horizontal and vertical scale-bars represent 20 ms and 1.0 nA, respectively. iii. Recordings from adult DRG neurons show a similar effect as early post-natal DRGs in which the P2/P1 ratio was enhanced in β-arr2-/- vs β-arr2+/+ neurons. This effect was enhanced by GTP-γ-S. ***p < 0.0001 vs β-arr2+/+., * p < 0.05 vs β-arr2+/+, #p < 0.05 vs untreated β-arr2-/- recordings.
Figure 2
Figure 2
Constitutive coupling of the μ-receptor with VDCC in β-arr2-/- but not β-arr2+/+ DRG neurons. A Constitutive coupling of GPCRs to VDCCs can be reversed by inverse agonists but not neutral antagonists. Accordingly the enhanced facilitation ratio in β-arr2-/- vs β-arr2+/+ neurons, shown in Figure 1 and reproduced here for comparison, was inhibited by naloxone (Nal), naltrexone (Ntx), but not by 6α-naloxol, 6β-naloxol (6α-Nal, 6β-Nal), 6β-naltrexol, (6β-Ntx), or by the combination of naloxone with 6α-naloxol (6α-Nal+Nal). These ligands had no effect in β-arr2+/+ neurons. The upper panels show exemplar traces generated by the two-step protocol (scale-bars represent 20 ms (horizontal) and 0.5 nA (vertical) and the data from 12-22 cells are summarized in the underlying graph, with P2/P1 ratio on the ordinate and treatment on the abscissa. ***p < 0.0001 vs β-arr2+/+, *p < 0.05 vs β-arr2+/+, #p < 0.05 vs untreated β-arr2-/-. The concentration of each drug in μM is indicated in parentheses. B. A single-step voltage protocol was used to measure Ba2+ current amplitude in the presence (2) or absence (1) of naloxone, or 6α- and 6β-naloxol, as shown by the exemplar traces in the top panels. Naloxone and naltrexone increased the current amplitude in β-arr2-/- but not β-arr2+/+ neurons, whereas 6α-, 6β-naloxol or 6β-naltrexol, or the combination of naloxone with 6α-naloxol, had no effect on the current amplitude in either cell type. The upper panels show exemplar traces for all conditions. Scale-bars represent 20 ms (horizontal) and 0.5 nA (vertical) and the data for 7-12 cells are summarized in the underlying graph, inhibition (%) shown on the ordinate and treatment on the abscissa. *p < 0.05 vs β-arr2+/+. The concentration of each drug in μM is indicated in parentheses.
Figure 3
Figure 3
Increased basal analgesia in β-arr2-/- mice is reversed by μ receptor inverse agonists and unaffected by neutral antagonists. A. Using time to respond to tail withdrawal from 48°C water, β-arr2-/- mice showed a delayed withdrawal latency compared to β-arr2+/+ mice, (p < 0.05 β-arr2-/- vs β-arr2+/+). B. Naloxone (0.5 mg/kg), reduced the tail withdrawal latency when administered to β-arr2-/- mice 30 and 60 min after injection (#p < 0.05 vs untreated β-arr2-/-), but had no effect in β-arr2+/+ or β-arr2+/- mice. C, D and E. 6α- and 6β-naloxol, (1 mg and 10 mg/kg respectively) and the combination of 6α-naloxol (1 mg/kg) with naloxone (0.5 mg/kg), had no effect on the analgesic profile of the β-arr2-/- mice who continued to show an attenuated response compared with β-arr2+/+ mice (*p < 0.05, **p < 0.005 and ***p < 0.0001 vs β-arr2+/+). F. Similar to naloxone, naltrexone (0.5 mg/kg) reduced the increase in basal analgesia seen in β-arr2-/- mice to wild-type levels 30 min after the injection, but had no effect in β-arr2+/+ or β-arr2+/-mice. **p < 0.001 vs β-arr2+/+ and *p < 0.05 vs β-arr2+/-, #p < 0.05 vs untreated β-arr2-/- mice. G. In contrast, 6β-naltrexol (10 mg/kg) had no effect on the analgesic profile of β-arr2-/-, β-arr2+/-, or β-arr2+/+ mice. **p < 0.001 vs β-arr2+/+ and *p < 0.05 vs β-arr2+/+ mice
Figure 4
Figure 4
Constitutive activity of the μ receptor prolongs withdrawal from noxious heat but neither contributes to mechanical pain nor naloxone-mediated conditioned place aversion in β-arr2-/- mice. A. Similar to the tail-immersion assay, β-arr2-/- mice exhibit a delayed latency to respond in the Hargreaves test of thermal pain. This delay was reversed by naloxone (0.5 mg/kg). **p < 0.001 vs β-arr2+/+, #p < 0.05 vs untreated β-arr2-/- mice. B. However, mechanical pain, as assessed by the response to von Frey filaments, was unaffected by the absence of β-arr2. C Naloxone conditioned place preference is similarly not affected by the absence of β-arr2. After 3 days of conditioning, the aversive effect of naloxone resulted in less time spent in the naloxone-paired chamber for both β-arr2+/+ and β-arr2-/- mice (F2,34 = 4.27, p < 0.05). "Habituation" represents the time spent in the naloxone-paired chamber prior to conditioning. "Test" represents the time spent in the naloxone-paired chamber 24 h after the last of the three conditioning sessions. Numbers on the abscissa represent doses (mg/kg) of naloxone administered.
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