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. 2012 May 22;3(7):505–509. doi: 10.1021/cn3000394

The δ Opioid Receptor Agonist SNC80 Selectively Activates Heteromeric μ–δ Opioid Receptors

Matthew D Metcalf †, Ajay S Yekkirala †,‡, Michael D Powers †, Kelley F Kitto ‡,⊥, Carolyn A Fairbanks ‡,§,⊥, George L Wilcox ‡,⊥,∥, Philip S Portoghese †,‡,⊥,*
PMCID: PMC3399572  PMID: 22860219

Abstract

graphic file with name cn-2012-000394_0002.jpg

Coexpressed and colocalized μ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing μ–δ heteromers than in all other cell lines. Intrathecal SNC80 administration in μ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates μ–δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric μ–δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo.

Keywords: μ opioid receptor, δ opioid receptor, μ−δ heteromer, SNC80, knockout, antinociception

Compound SNC80 (Figure 1) has been employed as the prototypic nonpeptide, selective δ-opioid receptor agonist for nearly 20 years. The experimental techniques employed to define the δ-selectivity of SNC80 included radioligand binding, smooth muscle assays, and in vivo antagonism studies employing selective δ-antagonists.1−4 SNC80 has found broad recognition and adoption as a selective δ-agonist standard, occupying the previously vacant nonpeptide selective δ-agonist niche in the opioid pharmacological toolbox.3

Figure 1.

Figure 1

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Structure of SNC80.

However, in view of the discovery of opioid receptor heteromers as targets for ligands over the past decade, it is possible that opioid receptor heteromers may be activated by SNC80. In this regard, three heteromers, μ–δ,5 μ–κ,6 and κ–δ,7 have been established in heterologous cell lines, (8) and selective ligands that target these heteromers have been identified for each of the above heteromers.9−11 Moreover, heteromeric μ–δ receptors have been detected using selective antibodies in both cultured cells and DRG neurons.12 Thus, δ-receptors organized as heteromers could be relevant in the action of SNC80.

In this regard, SNC80-treated μ-opioid receptor knockout (μ-KO) mice have been found to be without effect in a visceral antiwrithing test.13 Also, SNC80 produced an antihyperalgesic effect in WT mice but lacked this effect in μ-KO mice,14 an effect similar to a separate study in δ-KO mice.15 Additionally, in vitro studies on the effects of SNC80 on opioid receptors suggested the involvement of μ-opioid receptors on the activity of SNC80. These included the finding that colocalized μ- and δ-opioid receptors in large and small dorsal root ganglion (DRG) neurons (16) are cointernalized into the same subcellular compartment for lysosomal degradation upon treatment with SNC80.17 These reports point to the involvement of both μ- and δ-opioid receptors in the activity of SNC80.

In the present study, the opioid activity of SNC80 at μ–δ heteromers was investigated using both HEK-293 cells and knockout (μ-KO and δ-KO) studies in mice. The finding that both the in vitro and in vivo results indicate that SNC80 produces maximal efficacy only when both μ- and δ-opioid receptors are present suggests that selective activation of μ–δ heteromers may be involved in vivo.

The antinociceptive activity of SNC80 was determined in mice to investigate the identity of its receptor target in vivo. In this regard, we hypothesized that SNC80 would have reduced activity in knockout animals lacking one of the receptors due to inability to form the μ–δ heteromer. Wild-type, μ-KO, and δ-KO mice were employed to determine the contribution of both the μ- and δ-opioid receptors on SNC80-induced antinociception. SNC80 was administered via the intrathecal route of injection (i.t.) to mice and assessed in the warm water (52.2 °C) tail withdrawal assay (Figure 2), focusing attention on the spinal cord where in vivo data support receptor colocalization.16 A cumulative dosing schedule was chosen partly to address the limited solubility of SNC-80, given that the three highest doses totaling 300 nmol greatly exceeded the amount of the compound that could be dissolved in a single 5 μL intrathecal injection volume. In addition, multiple comparisons with previous noncumulative dosing experiments revealed no significant differences in outcomes, validating the approach. The graph (Figure 2) shows two different dose–response curves to illustrate the lack of effect of SNC80 at low doses in μ-KO mice. In WT (C57/129) mice, SNC80 has an ED50 = 49 nmol, 95% CI (43–56). The dose–response curve (calculated from the second, higher dosage curve) is right-shifted in a parallel manner 2.7-fold in μ-KO mice (ED50 = 131 nmol, 95% CI (111–153), Figure 2A). In the WT (C57BL/6) mice, SNC80 displayed an ED50 = 53.6 nmol, 95% CI (47.0–61.1), which was right-shifted 6.1-fold in δ-KO mice, ED50 = 327 nmol, 95% CI (216–494) (∼50% MPE, Figure 2B). The results indicate that the antinociceptive activity of SNC80 is reduced in either μ- or δ-opioid receptor knockout mice.

Figure 2.

Figure 2

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Antinociceptive effects of cumulative SNC80 doses in WT, μ-KO, and δ-KO mice. Dose–response curves for the cumulative spinal antinociceptive effect of SNC80 in the mouse tail flick assay. The y-axis represents the effect of the dose expressed in percentage of maximum possible effect (%MPE). (A) Comparison of WT (C57/129, □) and μ-KO (■) mice. (B) Comparison of WT (C57BL/6, △) and δ-KO (▲) mice. Graph A shows two cumulative dose–response curves (gray and black squares) for μ-KO mice.

SNC80 was assayed in heterologous HEK293 cells singly or coexpressing opioid receptors, the same coexpressing cell lines where functional heteromer was established by coimmunoprecipitation.8,18 The internal calcium release [Ca2+]i assay, previously established to determine opioid receptor activation,9,18,19 was used to determine the selective efficacy of SNC80-induced receptor activation. In this assay of opioid efficacy, the opioid receptor activation is shifted by the chimeric Δ6-Gqi4-myr protein (20) to a Gq response (release of intracellular calcium), which was measured fluorescently. Importantly, opioid efficacy in the [Ca2+]i assay has produced similar and consistent results when compared with opioid efficacy in the [35S]GTPγS assay providing a convenient, nonradiological measure of whole cell efficacy.9,19 SNC80 selectively activated μ–δ heteromer in HEK293 cells with an EC50 = 52.8 ± 27.8 nM (SEM) (Figure 3), with a mean peak ΔRFU effect of 746 ± 83 (SEM), n = 3 (12). Cells expressing other opioid receptors were substantially less potently activated. In this regard, SNC80 potency in cells expressing only δ-opioid receptors was at least 100-fold less than in cells that coexpressed μ–δ heteromers.

Figure 3.

Figure 3

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Activity of SNC80 on intracellular calcium ([Ca2+]i) release in HEK293 cells stably expressing opioid receptors with transiently expressed Δ6Gqi4-myr. The agonist-induced [Ca2+]i response (reaction with fluorescent dye) was measured in relative fluorescence units (RFU). The y-axis plots change in RFU as a measure of agonist-induced [Ca2+]i release (ΔRFU) ± SEM n ≥ 3 (12) except for κ- and μ–κ-expressing cells where n ≥ 2 (8). The x-axis plots the log of the molar concentration of SNC80.

Cells stably expressing the chimeric Gqi4 protein and transiently expressing μ- or δ-opioid receptors or coexpressing both μ- and δ-opioid receptors were also assayed as a control for possible variability in protein-expression related artifacts. These experiments displayed similar and consistent results (Figure S1, Supporting Information) compared with cells transiently expressing the chimeric G-protein.

The in vitro efficacy data derived from intact HEK-293 cells in the present study support the concept that the principal targets of SNC80 are heteromeric μ–δ receptors. As illustrated in Figure 3 and Figure S1, Supporting Information, SNC80 exhibits substantially greater activity in cells coexpressing μ- and δ-opioid receptors than in cells either singly expressing δ-opioid receptors or coexpressing δ- and κ-opioid receptors. Given the evidence for physical association of these receptors, the implication is that μ–δ heteromers are selectively activated by SNC80, particularly since the singly expressed δ-receptor does not produce potent activation. These findings, taken together with the reported low binding affinity of SNC80 for μ-receptors,4 suggest that it targets the δ-protomer of the μ–δ heteromer, thereby leading to activation of the complex.

Our in vivo data also suggest that SNC80-induced antinociception is produced via selective activation of μ–δ heteromer in the spinal cord.16,17 The rightward shift in the SNC80 dose–response curve in both μ-KO and δ-KO mice demonstrates that both μ- and δ-opioid receptor protomers in the heteromeric complex contribute to the antinociceptive activity of SNC80 in wild-type mice. This result is consistent with prior studies showing that SNC80-induced antinociception possesses both δ- and μ-opioid receptor-mediated21,22 components. The in vivo contribution of both receptors, as evidenced by reduced potency of SNC80 in both δ- and μ-knockout animals, is also one of three specific criteria for the establishment of the heteromeric complex established by IUPHAR guidelines.23

Additionally, our in vitro efficacy data demonstrating selective activation of μ–δ heteromers by SNC80 is further supported by trafficking and binding studies.17,24 That these trafficking studies revealed SNC80-induced cointernalization of μ- and δ-opioid receptors is consistent with activation of a μ–δ heteromer as the signaling unit.

The present study demonstrates that potent SNC80 activity requires the presence of μ- and δ-opioid receptors both in vitro and in vivo. Considered together, and in light of prior studies, the combined results presented here suggest that SNC80, in vivo, interacts selectively with the δ-protomer of the μ–δ heteromer, thereby leading to the activation of the heteromeric complex. While this conclusion does not invalidate previous research demonstrating that SNC80 is a δ-selective ligand in receptor homomer models, it strongly suggests the involvement of the μ–δ heteromer as a selective target in the signaling pathway. The implications of μ–δ receptor heteromers as a target for SNC80 suggest a more complex role for this ligand in vivo, given that μ-selective ligands (based on binding) also target μ–δ heteromers but via the μ-protomer.19 It therefore appears that the output of the agonist depends on which protomer in the μ–δ heteromer is targeted for activation. The fact that, unlike μ-agonists, SNC80 does not produce physical dependence (25) illustrates the complexity of the heteromeric system and suggests that different signaling pathways are dependent on the protomer that is activated in the μ–δ heteromeric complex.

Methods

Antinociception Assays

All experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. Drug stock solutions were dissolved in sterile water and diluted in sterile saline solution with the exception of SNC80, which was initially mixed with 1 equiv of tartaric acid before dissolution in sterile water. Antinociceptive effects of SNC-80 were assessed utilizing the warm water (52.5 °C) tail immersion assay.26 Animals employed were 129sv/C57BL6 mice (μ-opioid receptor WT), μ-KO (−/−) mice on a 129sv/C57BL6 background,27 C57BL/6 mice (δ-opioid receptor WT), and δ-KO (−/−) mice on a C57BL/6 background,28 all with ad libitum food access and a 12 hour light/dark cycle. Intrathecal (i.t.) drug administration was accomplished by direct lumbar puncture (29) as modified, see review in ref (30). A minimum of six mice were assayed at each dose tested, each mouse was used twice, and a total of 48 mice were used in the study. No antinociceptive differences were observed between male and female mice in any of the experiments. Tail flick (TF) latencies were obtained before drug administration to establish a baseline prior to drug treatment; shortly after baseline testing, the lowest dose of drug was injected intrathecally in 5 μL of vehicle, and TF latency was determined 7 min later. Immediately after testing, the subsequent dose of a cumulative dose–response curve was administered, and TF latency was determined 5 min later; three to four doses were thereby administered in rapid succession, and a cumulative dose–response curve was determined. Antagonists were administered 7 min prior to the initial agonist injection. A 12 s cutoff was employed in cases of no detectable response to avoid tissue damage.

Data Analysis and Statistics

Results are expressed as a percentage of maximum possible effect (% MPE) calculated using the following formula using Microsoft Excel (Redmond, WA). Data plotted were concentration–effect curves and ED50 calculated in Prism 5.0 (San Diego, CA). Data points lacking error bars include the error within the area of the point.

graphic file with name cn-2012-000394_m001.jpg

Intracellular Calcium Release Assay

The intracellular calcium release assay as described previously19 was used to determine the selectivity of SNC80 in activating opioid receptors with minor modifications. Briefly, HEK293 cells stably expressing opioid receptors were grown in DMEM (10% FBS, 1% P/S) in 10% CO2 atmosphere, transiently transfected with a chimeric G-protein Δ6-Gqi4-myr using lipofectamine 2000 (Invitrogen, Carlsbad, CA), plated into 96 well half area plates 24 h later, and assayed for intracellular calcium release 48 h after transfection.19 In the experiments involving HEK293 cells stably expressing the chimeric Δ6-Gqi4-myr protein, the procedure was identical except opioid receptor DNA was transiently transfected, and the amount of lipofectamine was doubled in the cotransfected cells. Assays employed the standard explorer FLIPR calcium dye kit in a Flexstation 3 apparatus both from Molecular Devices (Sunnydale, CA). SNC80 freebase was initially dissolved in DMSO. DMSO was employed such that the final DMSO concentration did not exceed 0.1% v/v at maximum, a concentration that did not significantly alter calcium flux from basal responses. To minimize experimental variability, all experiments were conducted at least three times with four internal repetitions, n ≥ 3 (12), except cells expressing κ- or μ–κ receptors where n ≥ 2 (8). Assay controls (blanks and standard ligands) were performed on every plate to eliminate technical variability and ensure uniform response.

Data Analysis and Statistics

The raw data were collected in SoftMax Pro (Molecular Devices) and analyzed in GraphPad Prism 5.0, GraphPad Software (San Diego, CA). Data were represented as the change in relative fluorescence units (ΔRFU) for each concentration and plotted as sigmoidal concentration–response curves using nonlinear regression with error bars representing SEM. Points without error bars have the error contained within the plotted point.

Materials

SNC80, morphine, DAMGO, and DPDPE were obtained from the NIDA drug supply program (National Institutes on Drug Abuse).

Acknowledgments

We would like to thank Mary Lunzer and Dan Schuster for technical assistance, Dr. John Pintar for generously providing the δ-KO mice, Dr. Horace Loh for the gift of the μ-KO mice, Dr. Evi Kostenis for the gift of the Δ6-Gqi4-myr DNA, and Dr. Jennifer Whistler for the gift of stable HEK 293 cells coexpressing opioid receptors.

Glossary

Abbreviations

[Ca2+]i

intracellular calcium release

DAMGO

[d-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin

DMEM

Dubleco’s modified eagle medium

DPDPE

[d-Pen2,5]-enkephalin

FBS

fetal bovine serum

HBSS

Hank’s balanced salt solution

HEK

human embryonic kidney cells

KO

homozygous (−/−) knockout

MPE

maximum percentage effect

P/S

penicillin/streptomycin

SNC80

4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxypheny)methyl]-N,N-diethylbenzamide

κ–δ

kappa–delta opioid receptor heteromer

μ–δ

mu–delta opioid receptor heteromer

μ–κ

mu–kappa opioid receptor heteromer

DRG

dorsal root ganglion

Supporting Information Available

Supplemental figure, methods employed in the collection of the supplemental data, discussion of absence of convulsions in these assays, and associated references. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

Portoghese, Metcalf, Yekkirala, Powers, Wilcox, and Fairbanks participated in research design. Metcalf, Kitto, and Powers conducted experiments. Metcalf performed data analysis. Portoghese, Metcalf, and Wilcox wrote or contributed to the writing of the manuscript.

This work was supported by the National Institutes of Health, National Institutes of Drug Abuse [Grants DA01533, DA07234, and DA15438].

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States

Supplementary Material

cn3000394_si_001.pdf (142.5KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

cn3000394_si_001.pdf (142.5KB, pdf)

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