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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Pain. 2010 Apr 24;150(1):17–21. doi: 10.1016/j.pain.2010.02.011

Multiple PKCε-dependent Mechanisms Mediating Mechanical Hyperalgesia

Elizabeth K Joseph 1, Jon D Levine 1
PMCID: PMC2916056  NIHMSID: NIHMS179313  PMID: 20456866

Abstract

We have recently implicated mitochondrial mechanisms in models of neuropathic and inflammatory pain, in some of which a role of protein kinase Cε (PKCε) has also been implicated. Since mitochondria contain several proteins that are targets of PKCε, we evaluated the role of mitochondrial mechanisms in mechanical hyperalgesia induced by proinflammatory cytokines that induce PKCε-dependent nociceptor sensitization, and by a direct activator of PKCε (ψεRACK), in the rat. Prostaglandin E2 (PGE2)-induced hyperalgesia is short lived in naïve rats, while it is prolonged in ψεRACK pre-treated rats, a phenomenon referred to as priming. Inhibitors of two closely-related mitochondrial functions, electron transport (complexes I-V) and oxidative stress (reactive oxygen species), attenuated mechanical hyperalgesia induced by intradermal injection of ψεRACK. In marked contrast, in a PKCε-dependent form of mechanical hyperalgesia induced by prostaglandin E2 (PGE2), inhibitors of mitochondrial function failed to attenuate hyperalgesia. These studies support the suggestion that at least two downstream signaling pathways can mediate the hyperalgesia induced by activating PKCε.

Keywords: Hyperalgesia, Mitochondria, Priming, Protein kinase Cε, Pain

Introduction

While PKCepsilon (PKCε) a novel protein kinase C (PKC) isoform, plays an important role in primary afferent nociceptor sensitization and mechanical hyperalgesia [28,3,43,40,10,42] mechanisms downstream of PKCε remain to be elucidated. The mitochondrion, an intracellular organelle to which PKCε can translocate [15,11,36] which contains proteins phosphorylated by PKCε such as the mitochondrial respiratory chain protein, cytochrome oxidase subunit IV (COX IV) [32,8,12], mitochondrial ATP-sensitive K+ channel [38,11], and glycogen synthatase kinase-3beta [7,29], has recently been implicated in mechanical hyperalgesia [24,25]. In the present study we evaluated the hypothesis that the mitochondrion is a downstream target of PKCε mediating the induction of mechanical hyperalgesia. In previous studies [26] we have shown that administration of carrageenan or ψεRACK can trigger long-lasting hypersensitivity of nociceptors to inflammatory cytokines. This phenomenon “hyperalgesic priming” was demonstrated to be PKCε dependent [35,21]. We studied the role of four key mitochondrial mechanisms, electron transport (complexes I-V), oxidative stress (reactive oxygen species, ROS), caspase signaling, and intracellular calcium mobilization [30,14,6], in mechanical hyperalgesia induced by inflammatory mediator G-protein coupled receptors that produce PKCε-dependent hyperalgesia [19,31] and by a more generalized activator of PKCε by pseudo-receptor for activated C kinase (ψεRACK), a direct activator of PKCε [13].

Experimental procedures

Animals

Male (220–300 g) Sprague–Dawley rats (Charles River, Hollister, CA) used in these experiments were housed in the Laboratory Animal Resource Center of the University of California, San Francisco, under a 12-h light/dark cycle. All experimental protocols were approved by the University of California, San Francisco, Institutional Animal Care and Use Committee, and conformed to NIH guidelines for the care and use of experimental animals. Effort was made to limit the number of animals used and their discomfort.

Drugs: Preparation and administration

The drugs used in these experiments were: rotenone (3 acetylphenyl N-(P-Tolyl) carbamate) a mitochondrial electron transport chain complex I (mETC-I) inhibitor; 3-NP (3 nitroproprionic acid), a mETC-II inhibitor; antimycin, a mETC-III inhibitor; sodium cyanide, a mETC-IV inhibitor; oligomycin, a mETC-V inhibitor; α-lipoic acid, an antioxidant, TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester), an intracellular calcium mobilization blocker, prostaglandin E2 (PGE2), and epinephrine (Sigma, St. Louis), ZVAD-FMK, a nonspecific caspase inhibitor (EMD Bioscience, San Diego), and ψεRACK, a selective PKCε activator [13] prepared by SynPep (Dublin, CA). Rotenone, 3-NP, antimycin, oligomycin, α-lipoic acid and ZVAD-FMK were dissolved in 10% DMSO, sodium cyanide, TMB-8, ψεRACK, and epinephrine were dissolved in physiological saline. PGE2 stock solution was prepared in 10% ethanol and diluted with physiological saline so that the final concentration of alcohol was < 1%. All compounds were administered intradermally (i.d.) on the dorsum of the hind paw in a volume of 5 μl, using a 30-gauge hypodermic needle connected to a 10-μl Hamilton syringe. When studying the effect of co-administration of the above inhibitors with other agents, the solutions were drawn up into the syringe, separated by a small air bubble, such that the inhibitor reached the paw prior to the other test agent. Paw withdrawal thresholds were measured at different time points (30 min and 4 hrs) except for time course determination, when it was measured up to 96 hrs after test agent administrations. Doses of test agents were selected based on the results of our previous studies [23,24].

Mechanical nociceptive testing

Mechanical nociceptive threshold was quantified by the Randall–Selitto paw pressure test [37], using an Analgesymeter (Stoelting, Chicago, IL) in which a force that increases linearly over time is applied to the dorsum of the rat’s hind paw. For testing, rats were lightly restrained in transparent cylinders with slits on the side that allowed access to the hind leg. All rats were acclimated to the testing procedure. Nociceptive threshold was defined as the force (in grams) at which the rat withdrew its paw. Paw-withdrawal threshold was defined as the mean of three readings. Each paw was treated as an independent measure and each experiment performed on a separate group of rats. All behavioral testing was done between 10:00 and 16:00 h.

Hyperalgesic priming was induced, as described previously [26,34,21]. In brief, the PKCε activator, ψεRACK (1 μg) was injected intradermally on the dorsum of a rat’s hind paw. ψεRACK-induced mechanical hyperalgesia was measured every hour for 6 hours on the first day and continued every day for four days. Five days later, when nociceptive threshold had returned to pre-ψεRACK treatment baseline, the nociceptive effect of PGE2 was evaluated. In this primed state PGE2 hyperalgesia develops PKCε dependence not present in the naïve control rat [4].

Statistics

Data are expressed as percentage decrease in nociceptive threshold, group data are presented as mean ± SEM, and analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc test. The accepted level for statistical significance was set at a p-value of 0.05. Separate groups of animals were used for each experiment.

Results

Time course of ψεRACK-, PGE2- and epinephrine-induced mechanical hyperalgesia

As previously demonstrated [5,26] intradermal injection of ψεRACK (1 μg), PGE2 (100 ng) and epinephrine (epi, 100 ng) induced a decrease in mechanical nociceptive threshold in control rats (Fig. 1A). The hyperalgesia induced by PGE2- and epinephrine- was short lived (< 4hr, Fig. 1A, both n = 6), while that induced by ψεRACK persisted for more than 24 hours (Fig. 1A, n = 6), returning to baseline by the 3rd to 4th day. In primed rats, which were previously treated with ψεRACK, administration of PGE2 (100 ng) now induced hyperalgesia that lasted for >4 hrs (Fig. 1B, n = 6). PGE2 and epinephrine were tested similarly and even when co-administered they failed to produce priming (data not shown).

Figure. 1. Time Course of ψεRACK, PGE2 and epinephrine hyperalgesia.

Figure. 1

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A. Time course of the mechanical hyperalgesia induced by intradermal injection of prostaglandin E2 (PGE2, 100 ng), epinephrine (epi, 100 ng) and a PKCε activator (ψεRACK, 1 μg) on the dorsum of the hind paw. All three agents tested, produced a decrease in mechanical nociceptive threshold measured at 30 min (all p < 0.001, n = 6/group), the mechanical hyperalgesia was short lived (< 4 hr) in PGE2-and epinephrine- treated rats, but prolonged (> 72 hr) in ψεRACK-treated rats.

B. Time course of the mechanical hyperalgesia induced by intradermal injection of prostaglandin E2 (PGE2, 100 ng) on the dorsum of the hind paw of rats treated 5 days previously with ψεRACK, 1 μg (i.e., primed). In ψεRACK primed rats, PGE 2 (100 ng)-induced peripheral mechanical hyperalgesia was significantly prolonged (> 24 hr, p < 0.001, n = 6).

Mitochondrial electron transport chain (mETC) complex inhibitors differentially affect ψεRACK-, PGE2- and epinephrine-induced hyperalgesia

To determine whether mitochondria play a role in mechanical hyperalgesia, we tested the effect of electron transport chain complex (mETC I-V) inhibitors on ψεRACK-PGE2- and epinephrine-induced hyperalgesia. Inhibitors of all five mETCs significantly attenuated ψεRACK-induced acute hyperalgesia (Fig. 2A, all, p < 0.001, n = 8/group) while all failed to inhibit PGE2- or epinephrine-induced hyperalgesia (Fig. 2B&C, all p = ns, n = 8/group). All mETC (I-V) inhibitors have been tested for their per se effect previously [24]. These compounds did not change the mechanical paw withdrawal thresholds of naïve rats at the doses employed.

Figure. 2. Effect of Mitochondrial inhibitors on ψεRACK, PGE2 and epinephrine-induced hyperalgesia.

Figure. 2

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A. Intradermal injection of inhibitors of mitochondrial electron complexes (I-V, each 5 μg) or ROS inhibitor α-lipoic acid (5 μg) together with ψεRACK (1 μg), significantly attenuated ψεRACK-induced hyperalgesia (all p < 0.001, n = 8/group). Non-selective caspase inhibitor, ZVAD-FMK and intracellular calcium mobilization blocker TMB-8 (both 5 μg, both p = ns; n = 8/group) did not affect ψεRACK-induced hyperalgesia.

B. Intradermal injection of inhibitors of mitochondrial electron complexes (I-V), ROS inhibitor α-lipoic acid, non-selective caspase inhibitor, ZVAD-FMK and intracellular calcium mobilization blocker TMB-8 did not attenuate PGE2 (100 ng)-induced hyperalgesia (all p = ns; n = 8/group).

C. Intradermal injection of inhibitors of mitochondrial electron complexes (I-V), non-selective caspase inhibitor, ZVAD-FMK and intracellular calcium mobilization blocker TMB-8 did not attenuate epinephrine (100 ng)-induced hyperalgesia (all p = ns; n = 8/group). However, ROS inhibitor α-lipoic acid significantly attenuated epinephrine-induced hyperalgesia (p < 0.001, n = 6).

Antioxidant differentially affect ψεRACK-, PGE2- and epinephrine-induced hyperalgesia

To evaluate the role of oxidative stress in mechanical hyperalgesia, we tested the effect of α-lipoic acid, an antioxidant, on ψεRACK-, PGE2- and epinephrine-induced mechanical hyperalgesia. α-lipoic acid markedly attenuate ψεRACK and slightly but significantly epinephrine-induced hyperalgesia (Fig. 2A&C, p < 0.001, for both ΨεRACK and epi, n = 8/for both groups), but failed to have any effect on PGE2-induced hyperalgesia (Fig. 2B, all p = ns; n = 8/group).

Caspase inhibitor does not inhibit ψεRACK-, PGE2- or epinephrine-induced hyperalgesia

To evaluate the role of apoptotic signaling pathways we tested the effect of a nonspecific caspase inhibitor Z-VAD-FMK on ψεRACK-, PGE2- and epinephrine-induced mechanical hyperalgesia. Z-VAD-FMK had no effect on mechanical hyperalgesia induced by ψεRACK-, PGE2- or epinephrine (Fig. 2 A,B&C, all p = ns; n = 8/group).

Calcium mobilization blocker does not inhibit ψεRACK-, PGE2- or epinephrine-induced mechanical hyperalgesia

To evaluate the role of calcium mobilization, we tested the effect of TMB-8, an intracellular calcium mobilization blocker, on ψεRACK-, PGE2 - and epinephrine-induced mechanical hyperalgesia. TMB-8 did not inhibit the mechanical hyperalgesia induced by ψεRACK, PGE2 or epinephrine (Fig. 2A,B&C, all p = ns; n = 8/group).

Role of mitochondria in PGE2 hyperalgesia after priming

After recovery from ψεRACK-induced mechanical hyperalgesia, PGE2 produces an enhanced (30 min) and PKCε-dependent prolonged (>4 hrs) hyperalgesia, a phenomenon referred to as hyperalgesic priming [4], in naίve control rats intradermal PGE2-induced hyperalgesia is mediated by protein kinase A (PKA) [4]. The mitochondrial electron transport chain complex (mETC I-V) inhibitors, α-lipoic acid, Z-VAD-FMK and TMB-8 were tested against PGE2-induced hyperalgesia in the primed state. In primed rats, none of these agents produced a significant reduction in the PGE2-induced hyperalgesia (Fig. 3A&B, all p = ns; n = 6/group).

Figure. 3. Effect of Mitochondrial inhibitors on PGE2-induced hyperalgesia in primed (previously treated with ψεRACK) rats.

Figure. 3

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A. Intradermal injection of mitochondrial electron complex inhibitors (I-V, each, 5 μg), ROS inhibitor α-lipoic acid, non-selective caspase inhibitor, ZVAD-FMK and intracellular calcium mobilization blocker TMB-8 (all 5 μg), did not attenuate PGE2 (100 ng)-induced hyperalgesia in ψεRACK-primed rats, when determined 30 min after the administration of PGE2 (all p = ns; n = 6/group).

B. Intradermal injection of inhibitors of mitochondrial electron complexes (I-V), ROS inhibitor α-lipoic acid, non-selective caspase inhibitor, ZVAD-FMK and intracellular calcium mobilization blocker TMB-8 (all n = 6/group) did not attenuate PGE2 (100 ng)-induced hyperalgesia in ψεRACK-primed rats, when determined 4 hr after the administration of PGE2 (all p = ns; n = 6/group).

Discussion

The mechanical hyperalgesia induced in several models of inflammatory [4,5,24,25], neuropathic [2,19,25] and generalized widespread pain [27] syndromes are, at least in part, PKCε dependent. Several molecules have been demonstrated to be upstream of PKCε in nociceptor function including G-protein coupled and death family cell surface receptors [3,20,24] and the second messengers Gi-protein [27] phospholipase Cβ3 [39,35,23,21], ceramide [23] and the guanine exchange factor, Epac [20]. However, the mechanisms downstream of PKCε in primary afferent nociceptors that mediate these pain syndromes, are largely unknown. The possibility that mitochondria play a role in transduction mechanisms in sensory neurons has been suggested by the finding that they are at especially high concentration in the peripheral terminals of presumed nociceptors in synovial [16] and corneal [17] afferents. Functional studies suggest that they may play a role in shaping nociceptor function [18,33]. We have recently demonstrated a role of mitochondrial mechanisms in a model of cancer chemotherapy (vincristine)-induced painful neuropathy in which mechanical hyperalgesia was PKCε – dependent [22,24]. Since it has been shown in other cells that PKCε can translocate to mitochondria [15,36], which contain proteins that are phosphorylated by PKCε including, mitochondrial respiratory chain proteins [32,8,12], in the present experiments we tested the hypothesis that mitochondrial mechanisms are downstream targets of PKCε mediating mechanical hyperalgesia.

While of the four major mitochondrial functions (i.e., electron transport and ATP synthesis, reactive oxygen species generation, caspase pathway signaling and intracellular calcium mobilization) [30,14,6], all have been implicated in mechanical hyperalgesia in one or more animal models, in the present studies we found that the hyperalgesia induced by direct activation of PKCε is mediated by only two of these mitochondrial mechanisms, the closely linked mitochondrial electron transport chain, and ROS generation. We did not detect a significant contribution from the other two mechanisms, caspase signaling and intracellular calcium mobilization, at doses found effective in other pain models [1,23].

While ψεRACK is likely able to activate PKCε in most if not all subcellular locations, it is known that activation of PKCε by different upstream signaling pathways causes it to translocate and therefore be activated at different sites in the cell (e.g., mitochondria, plasma membrane, Golgi, and juxta-nuclear region) [41,9,20,36]. Therefore, we also evaluated whether mitochondria are downstream of PKCε in receptor-activated signaling pathways involved in nociceptor sensitization. We chose to evaluate a model of chronic inflammatory pain, hyperalgesic priming, in which PKCε has been implicated in nociceptor sensitization and mechanical hyperalgesia [26,34,21]. In contrast to the effect of the more generalized activator of PKCε, hyperalgesia induced by PGE2 in primed paws was not dependent on any of the four major mitochondrial functions. While these data support a role for PKCε at a site in the cell other than that associated with the mitochondrial compartment in the cell, the location of the relevant pool of PKCε involved remains to be established. Of note, however, we have previously shown that a mediator that produces hyperalgesia by action at a G-protein coupled receptor (i.e., the β2-adrenergic receptor), which causes PKCε to translocate from the cytoplasm to plasma membrane [20], in the present study produced mitochondrial-independent hyperalgesia. Future studies will need to address both interactions between the mitochondrial mechanisms contributing to PKCε-induced hyperalgesia, as well as downstream targets of these mitochondrial mechanisms contributing to pain.

Acknowledgements

This work was supported by the NIH.

Footnotes

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Conflict of interest: Jon Levine is a consultant to KAI Pharmaceutical Inc., which is performing clinical trials of a PKCε-inhibitor for pain.

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