Protein kinase Cε is required for spinal analgesic synergy between delta opioid and alpha-2A adrenergic receptor agonist pairs

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.

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.

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.

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.

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.

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.

Immunoreactivity (ir) for PKCϵ in superficial dorsal horn of lumbar spinal cord relative to SP-ir, CGRP-ir, δOR-ir, or α_2AAR-ir. PKCϵ-ir is shown in red in the left panels, with SP-ir (A, B), CGRP-ir (C, D), α_2AAR-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 α_2AAR-ir in superficial dorsal horn. F, High-magnification images of lamina I/II showing in yellow that PKCϵ-ir and α_2AAR-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.