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. 2015 Dec;99:285-300.
doi: 10.1016/j.neuropharm.2015.08.010. Epub 2015 Aug 6.

Dual Allosteric Modulation of Opioid Antinociceptive Potency by α2A-adrenoceptors

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Dual Allosteric Modulation of Opioid Antinociceptive Potency by α2A-adrenoceptors

Anne-Julie Chabot-Doré et al. Neuropharmacology. .
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Abstract

Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations. Therefore, we investigated whether spinal opioid antinociception and opioid-adrenergic synergy were under allosteric control of the α2A-AR. Drugs were administered intrathecally in wild type (WT) and α2A-knock-out (KO) mice and antinociception was measured using the hot water tail immersion or substance P behavioral assays. The α2A-AR agonist clonidine was less effective in α2A-KO mice in both assays. The absence of the α2A-AR resulted in 10-70-fold increases in the antinociceptive potency of the opioid agonists morphine and DeltII. In contrast, neither morphine nor DeltII synergized with clonidine in α2A-KO mice, indicating that the α2AAR has both positive and negative modulatory effects on opioid antinociception. Depletion of descending adrenergic terminals with 6-OHDA resulted in a significant decrease in morphine efficacy in WT but not in α2A-KO mice, suggesting that endogenous norepinephrine acts through the α2A-AR to facilitate morphine antinociception. Based on these findings, we propose a model whereby ligand-occupied versus ligand-free α2A-AR produce distinct patterns of modulation of opioid receptor activation. In this model, agonist-occupied α2A-ARs potentiate opioid analgesia, while non-occupied α2A-ARs inhibit opioid analgesia. Exploiting such interactions between the two receptors could lead to the development of better pharmacological treatments for pain management.

Keywords: Analgesia; Morphine; Norepinephrine; Opioid receptor; Spinal cord; α(2-)adrenoceptor.

Figures

Figure 1
Figure 1. Comparison of thermal, mechanical and SP-induced nociceptive thresholds between WT and α 2A-KO mice
Tail flick latencies measured in the hot water (49°C) tail immersion assay (A) and paw withdrawal latencies measured with the radiant heat assay (B) were more rapid in α2A-KO mice. C) 50% mechanical threshold measured with von Frey filaments were similar on the hind paws of WT and α2A-KO mice. D) The number of nocifensive behaviors induced by administration of 15 ng of SP i.t. were not significantly different between WT and α2A-KO mice. Two-tailed unpaired t-test, ** p < 0.01, *** p < 0.0001.
Figure 2
Figure 2. Dose-response analysis of spinal clonidine-, morphine-, DeltII- and DAMGO-mediated antinociception in WT and α2A-KO mice
Dose-response curves were constructed for each drug administered i.t. in the hot water tail immersion assay (upper row) and the SP behavioral assay (middle row). A) Clonidine was efficacious in the tail immersion assay and the SP behavioral assay in WT mice but clonidine potency and efficacy were reduced in α2A-KO mice in both assays. B) Morphine was more potent in α2A-KO mice than in WT mice in both assays. C) DeltII was more potent in α2A-KO mice than in WT mice in both assays D) DAMGO was more potent in α2A-KO mice in the tail immersion assay but not in the SP behavioral assay. E) Clonidine was equally efficacious and potent in WT and α2C-KO mice. F) 100 nmol of SNC 80 was more effective in α2A-KO mice in the tail immersion assay compared to WT or vehiclecontrols. A–D) Dose-response curves generated with the SP behavioral assay in WT mice and the clonidine dose response curve in α2A-KO mice were published in Chabot-Doré et al. (2013) as part of a study conducted in parallel with this one. DeltII and DAMGO antinociception were measured with a cumulative dosing protocol in the tail immersion ED50 values are reported in Table 2. *p ≤ 0.01.
Figure 3
Figure 3. Synergistic interaction between clonidine and morphine in WT but not α2A-KO mice
Dose-response curves for spinal morphine, clonidine and their combination in WT mice in the hot water tail immersion assay (A) and the SP behavioral assay (D). B, E) Isobolographic analysis of the morphine and clonidine interaction in WT mice: the morphine ED50 value with lower CI is plotted on the y axis, and the clonidine ED50 value with lower CI is on the x axis. The measured experimental ED50 value (●) for the drug combination was lower than the theoretical additive ED50 value (), indicating that morphine and clonidine interacted in a synergistic manner (p < 0.05). In α2A-KO mice, since clonidine failed to reach 50% efficacy in the hot water tail immersion assay (C) or in the SP behavioral assay (F), isobolographic analysis was not performed. However, when clonidine was added to morphine in a 1:1 ratio, the ED50 values for morphine and clonidine were not significantly different than morphine alone (p < 0.05), showing that adding clonidine to morphine did not change its potency (F). The calculated experimental and theoretical ED50 values for the morphine and clonidine combinations are reported in Table 3. D, E were published in Chabot-Doré et al. (2013) as part of a study conducted in parallel with this one.
Figure 4
Figure 4. Synergistic interaction between clonidine and DeltII in WT but not α2A-KO mice
(A) Dose-response curves of spinal DeltII, clonidine and their combination in WT mice in the SP behavioral assay. (B) Isobolographic analysis of the DeltII and clonidine interaction in WT mice: the morphine ED50 value with lower CI is plotted along the y axis, and the clonidine ED50 value with lower CI along the x axis. The measured experimental ED50 value (●) for the drug combination was lower than the theoretical additive ED50 value (), indicating that DeltII and clonidine interact in a synergistic manner (p < 0.05). (C) In α2A-KO mice, since clonidine reached less than 50% efficacy isobolographic analysis was not performed. However, the doseresponse curves of DeltII and of clonidine+DeltII overlapped and ED50 values were not significantly different (p < 0.05), showing that adding clonidine to DeltII did not change its potency. The calculated experimental and theoretical ED50 values for the DeltII+clonidine combinations are reported in Table 3. A, B were published in Chabot-Doré et al. (2013) as part of a study conducted in parallel with this one.
Figure 5
Figure 5. Analysis of MOPr mRNA and protein expression in dorsal root ganglia (DRG) and spinal cords (SC) from WT and α2A-KO mice
Quantitative PCR analysis of the Oprm1 gene transcript from WT and α2A-KO DRG (A) and SC (B) showed no significant strain differences in MOPr mRNA levels. Relative expression was obtained by normalizing expression values to three internal housekeeping reference genes (Gapdh, Hptr1, Ubc) and then normalized with the WT value of that tissue as a reference. C) Representative western blot showing the 67 kDa MOPr-immunoreactive (ir) bands that were used to quantify MOPr levels in the spinal cord of WT and α2A-KO mice. D) Densitometry analysis of western blot MOPr-immunoreactive bands relative to GAPDH showed no strain difference between WT and α2A-KO.
Figure 6
Figure 6. Analysis of DOPr mRNA and [3H]-DeltII binding in WT and α2A-KO mice
Quantitative PCR analysis of the Oprd1 gene transcript from WT and α2A-KO DRG (A) and spinal cord (B) mRNA extracts showed no strain differences in DOPr mRNA expression. Relative expression was obtained by normalizing expression values to three internal house keeping reference genes (Gapdh, Hptr1, Ubc) and then normalized with the WT value of that tissue as a reference. C) Saturation ligand binding performed on spinal cord membrane protein extracts from WT and α2A-KO mice showing specific binding obtained by subtracting nonspecific from total [3H]-DeltII binding. The binding was concentration-dependent and no significant strain difference was detected. D) Competition of 1 nM [3H]-DeltII binding to spinal cord membranes by naltrindole was similar in WT and α2A-KO mice. CPM = counts per minute. DPM = disintregrations per minute.
Figure 7
Figure 7. Effect of selective elimination of spinal catecholaminergic nerve terminals on morphine antinociception and heat nociception
WT and α2A-KO mice were injected i.t. with either 6-OHDA or vehicle to eliminate catecholaminergic nerve fibers containing NE. A) Morphine dose-response curves were constructed using a cumulative dosage protocol. 6-OHDA reduced morphine response in WT mice, but not in α2A-KO mice. ED50 values are reported in Table 5. B) HPLC analysis of spinal content in norepinephrine (NE) and dopamine (DA) in ng/mg of spinal cord tissue. Vehicle-treated α2A-KO mice have lower NE and DA levels than WT mice. 6-OHDA treatment completely eliminated NE in both strains. C) Tail flick latency values were lower in 6-OHDAtreated WT mice compared to vehicle-treated mice. The 6-OHDA treatment had no effect on baseline latencies of α2A-KO mice. *** p < 0.001 ** p < 0.01.
Figure 8
Figure 8. Proposed model of allosteric regulation of opioid receptors by the α 2A-adrenoceptor
A) Schematic showing dose-response curves obtained for spinal morphine under different experimental conditions. In WT mice, morphine produces a dose-dependent antinociceptive effect (blue solid line), which is reduced in mice treated with 6-OHDA (blue dashed line) and potentiated by the addition of clonidine (purple solid line). In α2A-KO mice, morphine is more potent than in WT mice (red solid line) and neither the 6-OHDA treatment (red dashed line) nor the addition of clonidine (purple dashed line) shifted the morphine dose-response curve. B) In the proposed model, in WT mice, opioid receptors (OPr, green) are inhibited by the α2A-AR (red) in its ligand-free inactive state, which results in decreased opioid receptor antinociceptive output. When an agonist like clonidine (Clon) or norepinephrine (NE) activates α2A-AR, this inhibitory action on opioid receptors is removed, resulting in the synergism between the two agonistoccupied receptors. Under normal conditions, tonic NE release from descending noradrenergic fibers maintains an equilibrium state between ligand-free and agonist-occupied α2A-AR. When NE is depleted, the equilibrium is shifted towards the inactive and inhibitory α2A-AR, and when exogenous clonidine is administered, the equilibrium is shifted towards an active α2A-AR state, which disinhibits the opioid system. In α2A-KO mice, opioid receptors are not subjected to inhibition by the α2A-AR regardless of the presence or absence of α2A-AR ligands and are thus always fully activated.

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