Articles, Behavioral/Cognitive

Protein Kinase Cϵ Is Required for Spinal Analgesic Synergy between Delta Opioid and Alpha-2A Adrenergic Receptor Agonist Pairs

Daniel J. Schuster, Kelley F. Kitto, Aaron C. Overland, Robert O. Messing, Laura S. Stone, Carolyn A. Fairbanks and George L. Wilcox
Journal of Neuroscience 14 August 2013, 33 (33) 13538-13546; https://doi.org/10.1523/JNEUROSCI.4013-12.2013

Abstract

We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCϵ inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCϵ knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCϵ for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCϵ. Immunohistochemistry confirmed that PKCϵ could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCϵ was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCϵ is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs.

Introduction

Synergistic analgesic interactions between agonists of α2-adrenergic receptors (α2ARs) and opioid receptors (ORs) in rodents have been well documented (Hylden and Wilcox, 1983; Sullivan et al., 1987; Wilcox et al., 1987; Stone et al., 1997; Overland et al., 2009; Riedl et al., 2009) and polyanalgesic therapy using spinal coadministration of α2AR and OR agonists can reduce opioid requirements for pain management in the clinical setting (Förster and Rosenberg, 2004; Paech et al., 2004; Gregoretti et al., 2009). The α2A- and α2CAR subtypes, and all three OR subtypes (μ, δ, and κ) have been established as modulators of pain signaling at the spinal level (Stone et al., 1997; Fairbanks et al., 2002; Olave and Maxwell, 2002; Chakrabarti et al., 2010; Li et al., 2010). Certain α2AR agonists, such as clonidine (CLON) and brimonidine (BRIM), require the α2AAR-subtype for spinal analgesic efficacy and analgesic synergy with OR agonists (Stone et al., 1997; Fairbanks and Wilcox, 1999). Anatomical studies have shown that α2AARs are extensively colocalized with δORs in terminals of substance-P-expressing primary afferent fibers in rodents (Overland et al., 2009; Riedl et al., 2009), making these receptors a good pair for investigation of intracellular mechanisms of spinal opioid-adrenergic analgesic synergy.

Protein kinase C (PKC) has been established as an intracellular mediator of analgesic synergy between α2AARs and δORs both in vivo and in vitro. Inhibition of PKC blocks in vivo synergy between spinally coadministered CLON and deltorphin II (DELT) in the mouse tail flick test and blocks in vitro CLON-DELT synergistic inhibition of depolarization-evoked calcitonin gene-related peptide (CGRP) release from rat spinal cord slices (Overland et al., 2009). To determine whether a specific isoform of PKC mediates the synergistic interaction between α2AARs and δORs, we evaluated the ability of isoform-specific PKC inhibitor peptides to prevent spinal BRIM-DELT synergy. Inhibitors of the α, β (Ma et al., 2006), δ (Chen et al., 2001), and ϵ (Johnson et al., 1996) isoforms of PKC were tested, but only the PKCϵ inhibitor prevented BRIM-DELT synergy. To confirm and extend this finding, a battery of combinations of OR and AR agonists was tested for spinal analgesic synergy in mice genetically lacking PKCϵ. In addition, the PKCϵ activator peptide ψϵRACK (Dorn et al., 1999) was used to determine whether activation of PKCϵ is sufficient to enhance potency of either BRIM or DELT delivered singly. Finally, expression of PKCϵ in spinal cord and dorsal root ganglia (DRG) was evaluated using immunohistochemistry to determine whether PKCϵ is expressed in spinal terminals of peptidergic nociceptors, where analgesic synergy between α2AARs and δORs is thought to occur.

Materials and Methods

Animals.

Adult C57BL/6J PKCϵ wild-type (PKCϵ-WT), knock-out (PKCϵ-KO), and heterozygous mice of both sexes (20 ± 5 g) used for all experiments were bred from pairs of hybrid (50% C57BL/6J, 50% 129S4) mice heterozygous for the mutant PKCϵ gene (Khasar et al., 1999) and were maintained on a 12 h light/dark cycle with food and water available ad libitum to all animals. Heterozygous mice were used only for the PKC-isoform-specific peptide inhibitor experiments to conserve WT and KO mice and to make efficient use of all mice bred. All experiments were approved by the institutional animal care and use committee of the University of Minnesota.

Drug preparation and administration.

Agonists used were DELT (Tocris Bioscience), SNC80, BRIM (UK 14304), CLON, morphine sulfate (MOR), and endomorphin II (ENDO) (all from Sigma). The peptide activator of PKCϵ (ψϵRACK; HDAPIGYD; Dorn et al., 1999) and isoform-specific peptide inhibitors of PKC including the PKCα/β inhibitor FARKGALRQ (Ma et al., 2006), the PKCδ inhibitor SFNSYELGSL (Chen et al., 2001), and the PKCϵ inhibitor EAVSLKPT (Johnson et al., 1996), were purchased from Biomatik. The scrambled PKCϵ inhibitor (LSETKPAV) was from Anaspec. Peptide inhibitors or activators were administered 30 min before agonist administration and were preceded by an injection of 5 μl of distilled water to produce hypoosmotic shock and to facilitate passage of peptides through cell membranes (Khasar et al., 1999; Aley et al., 2000). BRIM was dissolved in a solution of 80% acetone, 6% DMSO, and normal saline to form a stock solution. SNC80 was dissolved with an equimolar amount of tartaric acid in saline. All other drug stocks were prepared in normal saline. All drugs were diluted from stock solution into sterile 0.9% saline and injected intrathecally in a volume of 5 μl in awake mice as described previously (Hylden and Wilcox, 1980).

Behavioral measures.

Thermal nociceptive responsiveness was assessed using the warm water (52.5°C) tail immersion assay as described previously (Janssen et al., 1963). Briefly, each animal was gently held wrapped in a cloth and the tail dipped into a controlled temperature water bath. Withdrawal latency was recorded as the amount of time that passed before a rapid movement of the tail and was not allowed to exceed 12 s. A baseline latency was recorded before drug administration and subsequent latencies were recorded 7 min after each dose immediately before the next dose. Each agonist or combination was administered sequentially approximately every 7 min in increasing doses to generate a cumulative dose-response curve, each mouse receiving no less than three and no more than four doses (Shin and Eisenach, 2003). Each dose-response curve was generated, with an n = 4–6 as indicated in each table or figure legend. For DELT, two dose-response curves were obtained on separate days to confirm the potency and efficacy of this drug in the PKCϵ mutant mice. These curves did not differ significantly in potency or efficacy and were combined for a total n = 12. This curve was then reused in the analysis for each combination tested. This allowed us to minimize the number of animals necessary to complete this study. Data are represented as percent maximum possible effect (%MPE) values, which were determined using the following equation: Embedded Image

Immunohistochemistry.

All animal tissues were fixed by intracardial perfusion as described previously (Vulchanova et al., 1998). Tissue sections were cut at 14 μm thickness and thaw mounted onto ProbeOn slides. Tissue sections were incubated for 1 h at room temperature in permeabilization buffer (TBS containing 0.2% Triton X-100, 0.2% Tween 20) and then incubated overnight at room temperature in primary antisera diluted in blocking buffer (TBS containing 0.2% Tween 20, 0.2% casein). Primary antibodies used were as follows: rabbit anti-PKCϵ (1:2000; Santa Cruz Biotechnology); guinea pig anti-substance P (SP; 1:1000; Neuromics); mouse anti-CGRP (1:2000; Research Biochemicals International); mouse anti-PKCϵ (1:300; Santa Cruz Biotechnology); rabbit anti-δOR (Dado et al., 1993; raised against amino acids 103–120 of δOR); and rabbit anti-α2AAR (Stone et al., 1998). After rinsing with TBS, sections were incubated for 1 h at room temperature with Cy3- or Cy5-conjugated secondary antisera (1:500; Jackson ImmunoResearch). Sections were rinsed again in TBS, dehydrated through a series of increasing ethanol concentrations followed by xylenes, and coverslipped using DPX mounting medium. Tissues were visualized using an Olympus FluoView 1000 BX2 upright confocal microscope and images were processed for color, contrast, and brightness using Adobe Photoshop.

Data analysis.

The ED50, in nanomoles with 95% confidence limits, of all agonists and combinations were calculated using the graded dose-response curve method of Tallarida and Murray (1987). Dose ratios for drug combinations were estimated based on comparison of ED50 values and dose-response curves and were chosen to approximate equi-effective doses. Isobolographic analyses were performed using the numerical method (Tallarida et al., 1989; Ossipov et al., 1997). Theoretical additive and observed combination ED50 values were compared statistically via the Student's t test with the JFlashCalc Pharmacological Calculations Program software package generously provided by Dr. Michael Ossipov (Department of Pharmacology, University of Arizona College of Medicine, Tucson, AZ). For all isobolograms, error bars for theoretical additive and observed combination ED50 values represent the vector sum of vertical and horizontal confidence limits.

Results

Selective inhibition of PKCϵ prevents analgesic synergy between BRIM and DELT

To determine whether a specific isoform of PKC is necessary for spinal analgesic synergy between an α2AAR agonist and a δOR agonist, we evaluated whether intrathecal pretreatment with various isoform-specific peptide inhibitors of PKC could prevent synergy between spinally delivered BRIM and DELT in the warm-water tail immersion assay. Isobolographic analysis revealed that pretreatment with 6 nmol of either a PKCα/β dual isoform inhibitor (FARKGALRQ; Ma et al., 2006) or a PKCδ inhibitor (SFNSYELGSL; Chen et al., 2001) had no effect on analgesic potency or efficacy of BRIM and DELT coadministered in approximately equi-effective doses; however, pretreatment with a PKCϵ inhibitor (EAVSLKPT; Johnson et al., 1996) significantly reduced potency of the BRIM-DELT combination such that the interaction was no longer synergistic (Fig. 1, Table 1). In addition, a scrambled version of the PKCϵ inhibitor (LSETKPAV) had no effect on potency of codelivered BRIM and DELT (Fig. 1, Table 1). These results indicate that of the four isoforms evaluated, only the ϵ isoform of PKC is involved in spinal analgesic synergy between agonist combinations acting at α2AARs and δORs. Numerical details for all statistical analyses are reported in Table 1.

Figure 1.
Figure 1.

Selective inhibition of PKCϵ, but not PKCα, PKCβ, or PKCδ, abolishes spinal analgesic synergy between BRIM and DELT. A, Dose-response curves for DELT (●), BRIM (■), and 1:1 combinations of both drugs with a pretreatment of one of the following: saline (□), PKCα/β inhibitor (FARKGALRQ; ▾), PKCδ inhibitor (SFNSYELGSL; ▵), PKCϵ inhibitor (EAVSLKPT; ♢), or scrambled PKCϵ inhibitor (LSETKPAV; ♦). All pretreatments were administered 30 min before BRIM and DELT. Error bars for each data point represent SEM. B–F, Isobolograms showing DELT dose and ED50 (■) on the x-axis, BRIM dose and ED50 (■) on the y-axis, theoretical additive ED50 for a 1:1 combination (○ on line of theoretical additivity), and observed combined ED50 (●). Error bars represent 95% confidence limits. B, Isobologram for BRIM and DELT after saline pretreatment illustrating a synergistic interaction. C, Isobologram for BRIM and DELT after pretreatment with a PKCα/β inhibitor (6 nmol, i.t.) illustrating a synergistic interaction. D, Isobologram for DELT and BRIM after pretreatment with a PKCϵ inhibitor (6 nmol, i.t.) illustrating a synergistic interaction. E, Isobologram for DELT and BRIM after pretreatment with a PKCϵ inhibitor (6 nmol, i.t.) illustrating an additive interaction. F, Isobologram for DELT and BRIM after pretreatment with a scrambled PKCε inhibitor (6 nmol, i.t.) illustrating a synergistic interaction. F, Isobologram for DELT and BRIM after pretreatment with a scrambled PKCε inhibitor (6 nmol, i.t.) illustrating a synergistic interaction. ED50 values for dose-response curves and p values for interactions can be found in Table 1.

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Table 1.

Interactions between BRIM and DELT in the presence or absence of PKC isoform-specific peptide inhibitors

Spinal analgesic synergy between δOR and α2AAR agonists requires PKCϵ

To confirm that PKCϵ plays a role in spinal analgesic synergy between α2AARs and δORs, various α2AAR and δOR agonists were administered intrathecally to PKCϵ-KO and PKCϵ-WT littermates and the effect of each single drug and each drug combination was evaluated using the warm-water tail immersion assay. Each of the δOR agonists, DELT and SNC80, was coadministered intrathecally in approximately equi-effective doses with each of the α2AAR agonists CLON and BRIM. All agonists produced comparable antinociception in both PKCϵ-WT and PKCϵ-KO mice when administered alone (Table 2). All four combinations of an α2AAR agonist and a δOR agonist produced analgesic synergy in PKCϵ-WT mice; however, none of these combinations synergized in PKCϵ-KO mice (Table 2). Dose-response curves and isobolograms for the BRIM + DELT (1:1) combination are shown in Figure 2 as representative examples of the synergistic interaction between the α2AAR and δOR agonist combinations in WT but not in PKCϵ-KO mice. These data indicate that spinal analgesic synergy between α2AARs and δORs requires PKCϵ. Numerical details for all statistical analyses are reported in Table 2.

View this table:
Table 2.

δOR and α2AAR agonist combinations in PKCϵ-WT and PKCϵ-KO mice

Figure 2.
Figure 2.

BRIM and DELT require PKCϵ for spinal analgesic synergy. A, C, Dose-response curves for BRIM (■), DELT (●), and a 1:1 combination of the two (□/○) in PKCϵ-WT (A) and PKCϵ-KO (C) mice. Error bars for each data point represent SEM. B, D, Isobolograms showing DELT dose and ED50 (■) on the x-axis, BRIM dose and ED50 (■) on the y-axis, theoretical additive ED50 for a 1:1 combination (○ on line of theoretical additivity), and observed combined ED50 (●). Error bars represent 95% confidence limits. B, BRIM and DELT produce analgesic synergy at the spinal level in PKCϵ-WT mice. D, The combination of BRIM and DELT is additive in PKCϵ-KO mice. ED50 values for dose-response curves and p values for interactions can be found in Table 2.

Analgesic synergy between μOR and α2AR agonists does not require PKCϵ

To determine whether PKCϵ is necessary for analgesic synergy between α2ARs and μORs, each of the μOR agonists, MOR and ENDO, was coadministered intrathecally in approximately equi-effective doses with CLON or BRIM and tested using the warm-water tail immersion assay. Both MOR and ENDO produced antinociception that was comparable between PKCϵ-WT and PKCϵ-KO mice (Table 3). All four combinations of α2AAR and μOR agonists produced analgesic synergy in both PKCϵ-WT and PKCϵ-KO mice (Table 3). Dose-response curves and isobolograms for the BRIM + MOR (1:1) combination shown in Figure 3 indicate that PKCϵ is not required for synergy between α2AARs and μORs. These data suggest that synergistic interactions between α2AARs and μORs occur through different mechanisms than those mediating synergy between α2AARs and δORs and are independent of PKCϵ. Numerical details for all statistical analyses are included in Table 3.

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Table 3.

μOR and α2AAR agonist combinations in PKCϵ-WT and PKCϵ-KO mice

Figure 3.
Figure 3.

BRIM and MOR do not require PKCϵ for spinal analgesic synergy. A, C, Dose-response curves for BRIM (■), MOR (●), and a 1:1 combination of the two (□/○) in PKCϵ-WT (A) and -KO (C) mice. Error bars for each data point represent SEM. B, D, Isobolograms showing MOR dose and ED50 (■) on the x-axis, BRIM dose and ED50 (■) on the y-axis, theoretical additive ED50 for a 1:1 combination (○ on line of theoretical additivity), and observed combined ED50 (●). Error bars represent 95% confidence limits. B, BRIM and MOR produce analgesic synergy at the spinal level in PKCϵ-WT mice. D, The combination of BRIM and MOR remains synergistic in PKCϵ-KO mice. ED50 values for dose-response curves and p values for interactions can be found in Table 3.

Activation of PKCϵ does not alter potency or efficacy of BRIM or DELT delivered singly.

To determine whether activation of PKCϵ is sufficient to enhance potency of α2AAR or δOR agonists given singly, we preceded BRIM or DELT dosing with a pretreatment of the PKCϵ activator ψϵRACK (Dorn et al., 1999). As expected, intrathecal delivery of ψϵRACK caused significant hyperalgesia (Joseph and Levine, 2010). This hyperalgesia was dose dependent (data not shown) and peaked at 30 min after delivery (Fig. 4). Pretreatment with ψϵRACK had no effect on the potency or efficacy of BRIM or DELT (Fig. 4), indicating that activation of PKCϵ is not sufficient to enhance potency of these agonists when they are delivered alone.

Figure 4.
Figure 4.

Activation of PKCϵ by intrathecal ψϵRACK does not significantly alter potency or efficacy of BRIM or DELT. A, Time course of tail-flick latencies after intrathecal delivery of ψϵRACK (30 nmol, i.t.) revealed significant hyperalgesia 30 min after injection (*significantly different from baseline, p < 0.01, one-way ANOVA with Bonferroni post hoc analysis). B, Pretreatment with ψϵRACK (30 nmol, 30 min) did not change potency or efficacy of DELT. C, Pretreatment with ψϵRACK (30 nmol, 30 min) did not change potency or efficacy of BRIM.

Immunohistochemical evaluation of localization of PKCϵ in spinal cord and dorsal root ganglion

To determine whether PKCϵ is located in peptidergic primary afferent nociceptors, where spinal analgesic synergy between δORs and α2AARs is thought to occur, sections of spinal cord and lumbar DRG were colabeled with antibodies directed against PKCϵ and each of the following: SP, CGRP, α2AAR, and δOR. Similar to previous reports (Khasar et al., 1999; Hucho et al., 2005; Wu et al., 2012), PKCϵ-immunoreactivity (ir) was observed in a large majority of DRG neurons (Fig. 5), as well is in laminae I and II of the superficial spinal cord dorsal horn (Fig. 6). As expected, PKCϵ-ir was not present in spinal cord or DRG of PKCϵ-KO mice (data not shown). In DRG, a subpopulation of CGRP- and SP-expressing neurons also displayed PKCϵ-ir (Fig. 5). In superficial dorsal horn, PKCϵ-ir was found in a portion of puncta displaying CGRP-ir or SP-ir (Fig. 6). In addition, we found that PKCϵ-ir partially colocalized with both α2AAR-ir and δOR-ir in the superficial dorsal horn (Fig. 6). This pattern of labeling indicates that PKCϵ is found in the location consistent with its role in mediating synergistic interactions between α2AARs and δORs. Although the pattern of labeling in superficial dorsal horn appears typical of that associated with incoming primary afferent terminals, closer examination at higher magnification revealed that PKCϵ-ir can also be found in neuronal cell bodies intrinsic to laminae I and II of the spinal cord, as evidenced by colocalization of PKCϵ-ir with ir for the neuronal label NeuN (data not shown). These results indicate that PKCϵ is expressed by multiple types of neurons involved in nociceptive processing at the spinal level.

Figure 5.
Figure 5.

Colocalization of immunoreactivity (ir) for PKCϵ and the neuropeptides SP and CGRP in lumbar DRG. PKCϵ-ir is shown in red in the left panels (A, D), with either SP-ir (B) or CGRP-ir (E) in green in the middle panels and overlaid images in the right panels illustrating that some neurons expressing PKCϵ also express SP or CGRP (C, F). A–C, Colabeling for PKCϵ-ir and SP-ir. D–F, Colabeling for PKCϵ-ir and CGRP-ir.

Figure 6.
Figure 6.

Immunoreactivity (ir) for PKCϵ in superficial dorsal horn of lumbar spinal cord relative to SP-ir, CGRP-ir, δOR-ir, or α2AAR-ir. PKCϵ-ir is shown in red in the left panels, with SP-ir (A, B), CGRP-ir (C, D), α2AAR-ir (E, F), or δOR-ir (G, H), in green in the middle panels and overlaid images in the right panels. A, Low-magnification images of superficial dorsal horn illustrating the pattern of PKCϵ-ir and SP-ir in this tissue section. B, High-magnification images of lamina I/II showing in yellow that PKCϵ-ir and SP-ir partially colocalize in spinal terminals of primary afferent neurons. C, Low-magnification images of PKCϵ-ir and CGRP-ir in superficial dorsal horn. D, High-magnification images of lamina I/II showing in yellow that PKCϵ-ir and CGRP-ir partially colocalize in spinal terminals of primary afferent neurons. E, Low-magnification images of PKCϵ-ir and α2AAR-ir in superficial dorsal horn. F, High-magnification images of lamina I/II showing in yellow that PKCϵ-ir and α2AAR-ir partially colocalize in spinal terminals. G, Low-magnification images of PKCϵ-ir and δOR-ir in superficial dorsal horn. H, High-magnification images of lamina I/II showing in yellow that PKCϵ-ir and δOR-ir partially colocalize in spinal terminals.

Discussion

The present study demonstrates that PKCϵ is necessary for synergistic analgesic interactions between α2AAR and δOR agonists at the spinal level in vivo. In contrast, other combinations that produce spinal analgesic synergy, such as those involving α2ARs and μORs, do not require PKCϵ. Because previous reports have placed α2AARs and δORs on the spinal terminals of peptidergic primary afferent neurons (Riedl et al., 2009) and shown that α2AAR and δOR agonists synergistically inhibit CGRP release from spinal cord slices (Overland et al., 2009) and spinal synaptosomes (Riedl et al., 2009), our behavioral data are consistent with the observations that PKCϵ colocalizes with α2AARs, δORs, SP, and CGRP. Immunolabeling of δORs and subsequent identification of δOR localization within either peptidergic or nonpeptidergic subpopulations of sensory neurons remains controversial. Considering this controversy, we believe that the functional evidence linking δOR agonists to inhibition of peptide release from primary afferents (Bao et al., 2003; Kondo et al., 2005; Wang et al., 2010; Beaudry et al., 2011) and the demonstrated involvement of PKC in the synergistic inhibition of CGRP release by coadministered α2AAR and δOR agonists in spinal cord (Overland et al., 2009) strongly support colocalization of δORs and α2AARs in peptidergic primary afferents. Factoring in the observed colocalization of PKCϵ with SP and CGRP in dorsal horn presented here strengthens our inference that PKCϵ mediates the synergistic interaction between α2AARs and δORs within peptidergic terminals.

One caveat common to most in vivo pharmacological studies, including this one, is that agonists often have activity at receptors other than their primary target. The fact that four synergistic combinations thought to involve primarily α2AARs and δORs (CLON-DELT, BRIM-DELT, CLON-SNC80, BRIM-SNC80) all required PKCϵ strengthens the evidence that these two receptor subtypes are responsible for analgesic synergy between these compounds. Although opioid-adrenergic analgesic synergy is well documented at the spinal level, there has been little investigation into the mechanisms underlying these interactions. The present study indicates that different combinations of an OR and an AR agonist can produce analgesic synergy through different mechanisms. The combination of: (1) the colocalization of α2AARs and δORs on peptidergic primary afferent nociceptors (Stone et al., 1998; Overland et al., 2009; Riedl et al., 2009), (2) the present result that PKCϵ can also be found in these neurons, and (3) the observation that four different combinations of α2AAR and δOR agonists all required PKCϵ for spinal analgesic synergy suggests that synergy between these receptors occurs in primary afferent terminals. Several studies indicate that signaling mechanisms initiated by agonist binding at α2AARs and/or δORs could lead to activation of PKCϵ. Agonist binding at either α2AARs or δORs induces release of intracellular calcium stores via Gαi-associated Gβ/γ activation of phospholipase C (PLC) (Dorn et al., 1997; Yoon et al., 1999). Therefore, these receptors could produce activation of PKCϵ through increased production of diacylglycerol via the same pathway. In fact, a link has been established between Gi signaling and activation of PKCϵ specifically in primary afferent neurons (Dina et al., 2009). It is also possible that α2AARs and/or δORs signal to PKCϵ through the more typical Gαq to PLC pathway. Delta ORs have been shown to couple to several G-proteins, including Gαq (Standifer et al., 1996; Sánchez-Blázquez and Garzon, 1998) and Gα16 (Lee et al., 1998; Chan et al., 2003), both of which signal to PLC. Alternatively, it is possible that only baseline activity of PKCϵ is needed for the observed interaction between receptors, rather than receptor-mediated activation. Although either α2AARs or δORs may activate PKCϵ, it is clear from our data showing similar efficacy of AR and OR agonists delivered individually in WT versus PKCϵ-KO mice that PKCϵ is not involved in the antinociceptive activity of these drugs when administered alone.

The effectors that PKCϵ acts upon to produce the synergistic interaction between α2AARs and δORs remain unknown. The failure of the PKCϵ activator ψϵRACK to increase the potency of either agonist given alone suggests that simple receptor phosphorylation by PKCϵ is not the only event necessary to increase agonist potency. Nonetheless, one mechanism by which PKCϵ may exert its effects is through phosphorylation of α2AARs or δORs themselves. Modification of these receptors may induce greater functional capability by increasing activity with inhibitory G-proteins, making them more responsive to agonists or altering the way they interact with each other. Indeed, there is evidence that PKC can induce functional competence of δORs trigeminal ganglion cultures directly (Patwardhan et al., 2005), as well as in vivo in the periphery (Rowan et al., 2009), although it is unknown which isoform of PKC is responsible for the observed effects. Additional evidence suggests that lack of the ϵ isoform of PKC decreases stimulus-induced release of CGRP (Sweitzer et al., 2004). Because primary afferent plasma membrane δOR expression is thought to be limited at a basal state and augmented by trafficking of large dense-core vesicles under certain stimulus conditions (Zhao et al., 2011), including δOR agonist exposure (Bao et al., 2003; Overland et al., 2009), loss of PKCϵ may result in decreased surface and/or functional availability of δORs. Further studies are needed to determine what specific role, if any, PKCϵ plays in the modulation of trafficking and function of δORs or α2AARs.

Because PKCϵ is found in the majority of DRG neurons, it is not surprising that it is expressed in several subpopulations of sensory neurons (Hucho et al., 2005; Ferrari et al., 2010; Joseph and Levine, 2010). Studies by Ferrari et al. (2010) and Joseph and Levine (2010) have shown that PKCϵ mediates hyperalgesic priming in the peripheral terminals of isolectin-B4-binding neurons. The present study indicates a different role for PKCϵ in DRG by showing that in the central terminals of some peptidergic afferents, it can be involved in enhanced antinociception upon activation of the appropriate receptor combination. We also report that PKCϵ can be found not only in primary afferent neurons, but also intrinsic spinal neurons located in the superficial dorsal horn (laminae I and II). The complexity of the involvement of PKCϵ in nociceptive processing is becoming increasingly apparent because it clearly plays multiple roles across several neuronal subtypes at both the peripheral and spinal levels.

The data presented here show that synergistic analgesic interactions between adrenergic and opioid agonists at the spinal level occur through different mechanisms depending on the anatomical relationship between the targeted receptor subtypes. Specifically, synergistic interactions between agonist combinations acting at α2AARs and δORs require PKCϵ and likely occur in the spinal terminals of peptidergic nociceptors. Conversely, synergistic interactions between agonists acting at receptors thought to be located on different neurons, such as α2ARs and μORs, do not require PKCϵ. PKCϵ is widespread in the spinal nociceptive system and has multiple known pro-nociceptive actions. In contrast, the present study shows that coactivation of α2AARs and δORs leads to involvement of PKCϵ in antinociceptive synergy. Our results have revealed a new role for PKCϵ in spinal nociceptive processing and furthered our understanding of synergistic analgesic interactions between α2ARs and ORs at the spinal level.

Footnotes

  • This work was supported by the National Institutes of Health (National Institute on Drug Abuse Grant #R01 DA015438 to G.L.W. and National Institute of Neurological Disorders and Stroke Grants #P01 NS053709 to R.O.M. and #F31 NS063634 to D.J.S.). We thank Steven A. Schnell and Drs. Maureen Riedl and Lucy Vulchanova for discussions on immunohistochemical methods and results and Galina Kalyuzhnaya for technical assistance.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to George L. Wilcox, PhD, Departments of Neuroscience, Pharmacology and Dermatology, 4–136 MCB, 420 Washington Ave S, Minneapolis, MN 55455. george{at}umn.edu

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Protein Kinase Cϵ Is Required for Spinal Analgesic Synergy between Delta Opioid and Alpha-2A Adrenergic Receptor Agonist Pairs
Daniel J. Schuster, Kelley F. Kitto, Aaron C. Overland, Robert O. Messing, Laura S. Stone, Carolyn A. Fairbanks, George L. Wilcox
Journal of Neuroscience 14 August 2013, 33 (33) 13538-13546; DOI: 10.1523/JNEUROSCI.4013-12.2013
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