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The Journal of Neuroscience, September 1, 2001, 21(17):6933-6939

Nociceptor Sensitization by Extracellular Signal-Regulated Kinases

K. O. Aley1, Annick Martin2, Thomas McMahon2, Janine Mok1, Jon D. Levine1, and Robert O. Messing2

1 Departments of Medicine and Oral Surgery, National Institutes of Health Pain Center at the University of California, San Francisco, San Francisco, California 94143, and the 2 Department of Neurology, Ernest Gallo Clinic and Research Center at the University of California, San Francisco, Emeryville, California 94608


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inflammatory pain, characterized by a decrease in mechanical nociceptive threshold (hyperalgesia), arises through actions of inflammatory mediators, many of which sensitize primary afferent nociceptors via G-protein-coupled receptors. Two signaling pathways, one involving protein kinase A (PKA) and one involving the epsilon isozyme of protein kinase C (PKCepsilon ), have been implicated in primary afferent nociceptor sensitization. Here we describe a third, independent pathway that involves activation of extracellular signal-regulated kinases (ERKs) 1 and 2. Epinephrine, which induces hyperalgesia by direct action at beta 2-adrenergic receptors on primary afferent nociceptors, stimulated phosphorylation of ERK1/2 in cultured rat dorsal root ganglion cells. This was inhibited by a beta 2-adrenergic receptor blocker and by an inhibitor of mitogen and extracellular signal-regulated kinase kinase (MEK), which phosphorylates and activates ERK1/2. Inhibitors of Gi/o-proteins, Ras farnesyltransferases, and MEK decreased epinephrine-induced hyper-algesia. In a similar manner, phosphorylation of ERK1/2 was also decreased by these inhibitors. Local injection of dominant active MEK produced hyperalgesia that was unaffected by PKA or PKCepsilon inhibitors. Conversely, hyperalgesia produced by agents that activate PKA or PKCepsilon was unaffected by MEK inhibitors. We conclude that a Ras-MEK-ERK1/2 cascade acts independent of PKA or PKCepsilon as a novel signaling pathway for the production of inflammatory pain. This pathway may present a target for a new class of analgesic agents.

Key words: extracellular signal-regulated kinase; mitogen-activated protein kinase; protein kinase Cepsilon ; protein kinase A; epinephrine; pain; hyperalgesia; nociceptor; Ras; beta -adrenergic receptor; mitogen and extracellular signal-regulated kinase kinase; G-protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Current evidence indicates that at least two signaling pathways mediate hyperalgesia produced by inflammatory agents. The inflammatory mediators prostaglandin E2 (PGE2), serotonin, and adenosine produce hyperalgesia through activation of protein kinase A (PKA) (Gold et al., 1996, 1998; Khasar et al., 1998a, 1999a), and this process is facilitated by nitric oxide (Aley et al., 1998; Chen and Levine, 1999). On the other hand, epinephrine, acting through beta 2-adrenergic receptors on primary afferent nociceptors, produces mechanical hyperalgesia in part through PKA but also through the epsilon isozyme of protein kinase C (PKC epsilon ) (Khasar et al., 1999a). PKCepsilon also contributes to bradykinin-induced sensitization of nociceptors to heat (Cesare et al., 1999).

PKA and PKCepsilon mediate nociceptor sensitization by modulating the activity of a tetrodotoxin-resistant sodium current that is sensitized by direct-acting hyperalgesic agents (Gold et al., 1996; Khasar et al., 1999a,b). We originally thought that PKA and PKCepsilon signaling pathways might converge at extracellular signal-regulated kinases 1 and 2 (ERK1/2), because ERK1/2 are modulated by PKA and PKCepsilon (Hundle et al., 1995; Vossler et al., 1997; Grewal et al., 2000). Moreover, beta 2-adrenergic receptors, like several other G-protein-coupled receptors, can activate ERKs (Daaka et al., 1997; Della Rocca et al., 1997; Wan and Huang, 1998; Maudsley et al., 2000; Schmitt and Stork, 2000). ERKs are mitogen-activated protein (MAP) kinases that mediate several cellular responses to mitogenic and differentiation signals (Lewis et al., 1998). They are activated by diverse extracellular stimuli, including several hormones and growth factors that activate G-protein-coupled receptors or receptor tyrosine kinases, leading to stimulation of Raf kinases, which phosphorylate and activate mitogen and extracellular signal-regulated kinase kinase (MEK). Activated MEK in turn phosphorylates and activates ERK1/2. PKA and cAMP can promote ERK activation via a Rap1-dependent pathway in neural cells, such as PC12 cells, that use B-Raf as the major Raf isoform (Ohtsuka et al., 1996; Vossler et al., 1997; Kawasaki et al., 1998; York et al., 1998; Grewal et al., 2000). In PC12 cells, PKCepsilon promotes ERK phosphorylation and activation by nerve growth factor (NGF) or epidermal growth factor (EGF) through an unknown mechanism (Hundle et al., 1995, 1997; Brodie et al., 1999). Thus, activation of ERKs in nociceptors could provide an important mechanism for convergence of PKA and PKCepsilon signaling pathways.

In this paper, we examined whether ERK activation is involved in pain signaling by examining epinephrine-treated rat dorsal root ganglion (DRG) neurons in culture and epinephrine-induced mechanical hyperalgesia in rats. We report that epinephrine activates ERKs in cultured DRG neurons and that a heterotrimeric Gi- or Go-protein, Ras, and MEK contribute to epinephrine-induced hyperalgesia, independent of PKCepsilon or PKA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Epinephrine, the selective beta 2-adrenergic receptor antagonist ICI 118,551, PGE2 pertussis toxin, epinephrine, and the isoprenylation inhibitor perillic acid were purchased from Sigma (St. Louis, MO). The general PKC inhibitor bisindoylmaleimideI (BIM), the PKA inhibitor H89, the MEK inhibitors U0126 and PD98059, mouse 2.5 S NGF, and the farensyltransferase inhibitor FTase I were from Calbiochem (La Jolla, CA). The Walsh inhibitor peptide (WIPTIDE) of PKA was purchased from Peninsula Laboratories (Belmont, CA). Anti-phospho-p42/44 MAP kinase (Thr202/Tyr204) antibody against the MEK-phosphorylated forms of ERK1/2 and anti-ERK1/2 antibody were purchased from New England Biolabs (Beverly, MA) or, where indicated, from Upstate Biotechnology (Lake Placid, NY). Dominant active and kinase inactive recombinant MEK1 were purchased from Upstate Biotechnology. A specific activator of PKCepsilon , psi epsilon RACK (receptor for activated C kinase), was a gift from D. Mochly-Rosen (Stanford University, Stanford, CA). A specific inhibitor of PKCepsilon , epsilon V1-2, was synthesized by SynPep (Danville, CA).

Cell culture. Dorsal root ganglia were collected from male adult Sprague Dawley rats (200 gm) obtained from Simonsen (Gilroy, CA) or from PKCepsilon null and wild-type C57BL/6J × 129 SvJae mice of the F2 generation (Khasar et al., 1999a). The cells were dissociated by treating ganglia with 0.125% collagenase P for 2 hr, followed by a trypsin solution (0.025% trypsin and 0.025% EDTA in HBSS) for 15 min. Trypsin was inactivated by adding 100 µg/ml soybean trypsin inhibitor and 2.5 mg/ml MgSO4. The cells were centrifuged at 300 × g for 5 min and resuspended in culture media containing minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum, 1× MEM vitamins, and 1000 U/ml each of penicillin and streptomycin. The culture was enriched for neurons by preplating on 100 mm culture dishes pretreated with 0.1 mg/ml poly-DL-ornithine in 15 mM sodium borate buffer. After culture for 15-20 hr, the loosely attached neuronal cells were collected and plated for 3 hr on six-well plates coated with 0.1 mg/ml poly-DL-ornithine and 1 mg/ml laminin.

Western analysis. After drug treatment, cells from neuron-enriched DRG cultures were collected and centrifuged at 300 × g for 5 min at 4°C. The pellets were resuspended in lysis buffer [50 mM Tris HCl, pH 7.4, 1% (v/v) NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, and 10 mM EDTA] and protease inhibitors (leupeptin and aprotinin at 40 µg/ml each, 25 µg/ml soybean trypsin inhibitor, and 1 mM PMSF) and phosphatase inhibitors (25 mM NaF, 1 mM Na3VO4, 40 mM beta  glycerophosphate, and 1 mM Na pyrophosphate). Proteins in 200 µg samples of cell lysates were separated by SDS-PAGE using 12% polyacrylamide gels. The proteins were electroblotted onto Hybond C nitrocellulose membranes, which were incubated in a blocking solution containing 5% nonfat dry milk dissolved in PBS-T (137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4, 8 mM NaHPO4, 0.5 mM MgCl2, and 0.9 mM CaCl2, pH 7.2, and 0.1% Tween 20). Blots were incubated with anti-phospho-p42/44 MAP kinase antibody (diluted 1:500 in blocking buffer) overnight at 4°C. Blots were rinsed three times in PBS-T and incubated in blocking buffer containing HRP-conjugated goat anti-rabbit IgG (diluted 1:1000; Boehringer Mannheim, Indianapolis, IN) for 1 hr at 27°C. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then stripped of antibodies by incubation in 200 mM NaOH for 20 min at 27°C. After three washes in PBS-T, blots were incubated with anti-ERK1/2 antibody (136 ng/ml) for 1 hr at 27°C. After three washes in PBS-T, blots were incubated with HRP-conjugated goat anti-rabbit IgG (diluted 1:1000; Boehringer Mannheim) for 1 hr at 27°C, and immunoreactive bands were visualized by enhanced chemiluminescence and autoradiography. Immunoreactive bands on autoradiograms were analyzed by scanning densitometry using a flatbed scanner and NIH Image version 1.62 (W. Rasband, National Institutes of Health, Bethesda, MD). Data were normalized by dividing values obtained for phospho-ERK1 and phospho-ERK2 immunoreactivity by the value obtained for total ERK1 immunoreactivity for each sample.

Immunofluorescence. Adult male Sprague Dawley rats were anesthetized with pentobarbital and transcardially perfused with PBS, followed by 4% paraformaldehyde (in PBS). DRGs were removed, post-fixed in 4% paraformaldehyde for 4 hr, treated with 30% sucrose (in PBS) for 24 hr, and then embedded in Tissue-Tek OCT. Cryosections (8 µm) were cut and stored at -20°C. Mounted DRG sections were allowed to thaw to room temperature. Sections were then incubated for 1 hr in blocking solution (PBS containing 5% normal donkey serum and 0.1% Triton X-100), overnight with anti-ERK1/2 (0.82 µg/ml; Upstate Biotechnology) and 1 hr with FITC-conjugated donkey anti-rabbit (7.5 µg/ml; Jackson ImmunoResearch, West Grove, PA). Both primary and secondary antibodies were diluted in 1.5% normal donkey serum, in PBS.

Animal housing. For behavioral studies, male Sprague Dawley rats (200-250 gm; Bantin-Kingman, Fremont, CA) were individually housed and maintained under a 12 hr light/dark cycle. The experimental rats were fed standard lab chow ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

Mechanical nociceptive threshold. The nociceptive flexion reflex (Randall-Selitto paw-withdrawal test) was quantified with a Basile Analgesymeter (Stoelting, Chicago, IL), which applies a linearly increasing mechanical force to the dorsum of the rat's hindpaw. The mechanical nociceptive threshold was defined as the force in grams at which the rat withdrew its paw. On the day of the test, animals were brought to the laboratory and allowed to remain in the cage for 10-15 min. They were allowed to crawl into individual cylindrical Perspex blocks and were lightly restrained there by closing both ends of the cylinder. The hindpaws of the rats were freed out of the cylinder through triangular slits on either side of the Perspex block, which allows easy access to the hindpaws during the test (Aley and Levine, 1999). The rats were allowed to acclimatize to the restrainer for 5-10 min, after which the hindpaws were exposed to the test stimulus. Three readings were taken at 5 min intervals, and their mean was considered the baseline threshold. After each drug administration, mechanical paw-withdrawal thresholds were determined again as the mean of three readings taken 20, 25, and 30 min after injection. The result was expressed as the percentage decrease in nociceptive threshold [(paw-withdrawal threshold after the drug - basal paw withdrawal threshold)/basal withdrawal threshold × 100].

Drugs for in vivo studies. Stock solutions (1 µg/µl) of BIM (in 10% dimethylsulfoxide) and epsilon V1-2 and WIPTIDE (in 0.9% saline) were stored at 20°C. Inhibitors were diluted with distilled water before intradermal injections into the paw using a 10 µl microsyringe (Hamilton, Reno, NV). Injections of peptides (1 µg/2.5 µl) were always preceded by injection of distilled water (2.5 µl) to produce hypo-osmotic shock. This was done to increase cell membrane permeability to these agents (Tsapis and Kepes, 1977; West and Huang, 1980; Taiwo and Levine, 1989; Khasar et al., 1995; Widdicombe et al., 1996). The dose of each protein kinase inhibitor was separated from the distilled water by an air bubble (<1 µl) so that the distilled water was injected into the paw first. Paw-withdrawal thresholds were measured again 10, 15, and 20 min after injecting the test agent. The mean of the paw-withdrawal thresholds obtained at these three times was then taken as the mechanical nociceptive threshold at the dose of the test agent used. The effect of each dose of a test agent was calculated as the percentage change from baseline.

Statistical analysis. The data are presented as mean ± SE values and were compared using the one way ANOVA, followed by Newman-Keuls, Tukey's, or Dunnett's post hoc tests, as noted. Differences between means were considered significant at p < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because of the recent evidence linking beta 2-adrenergic receptor stimulation to activation of ERKs in non-neuronal cells (Daaka et al., 1997; Della Rocca et al., 1997; Wan and Huang, 1998; Maudsley et al., 2000; Schmitt and Stork, 2000), we examined whether ERK1/2 are present in rat DRG neurons and are activated by epinephrine. Immunofluorescence staining of isolated DRG demonstrated ERK1/2 immunoreactivity in cell bodies of DRG neurons (Fig. 1A). To measure responses to epinephrine, we next examined DRG neurons in culture. In unstimulated neurons, there was a basal level of phospho-ERK immunoreactivity (Fig. 1B,C). This was increased by ~1.7-fold after incubation with 1 µM epinephrine. Epinephrine-evoked ERK phosphorylation was greatest after 5 min and gradually returned to basal levels after 60 min. The effect of epinephrine was dose-dependent and appeared to be maximal at a concentration of 1 µM (Fig. 1D). The maximal response to epinephrine was ~30-50% of the response observed after incubation with a maximally effective concentration of NGF (50 ng/ml) for 5 min (Fig. 1B).



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  		Figure 1.   Epinephrine stimulates ERK1/2 phosphorylation in DRG neurons. A, ERK1/2 immunoreactivity present in cell bodies of neurons in freshly isolated DRG. The top shows incubation with anti-ERK1/2, whereas the bottom shows loss of immunoreactivity after preincubation of antibody with excess of peptide antigen. Scale bar, 100 µm. B, DRG cultures were treated with epinephrine for the indicated times and then processed for analysis of phospho-ERK1/2 immunoreactivity by Western analysis. Some cells were treated instead with 50 ng/ml NGF for 5 min as a positive control. Blots were then stripped and probed with anti phospho-ERK1/2 antibody. Representative Western blot demonstrating NGF and epinephrine stimulation of ERK1/2 phosphorylation. C, Mean ± SE values (n = 7-23) for phospho-ERK1/2 immunoreactivity normalized to total ERK1/2 immunoreactivity. *p < 0.05 by ANOVA and Dunnett's multiple comparison test. D, Representative Western blot showing concentration dependence of epinephrine-induced ERK1/2 phosphorylation.

Epinephrine-induced stimulation of ERK phosphorylation was mediated by beta 2-adrenergic receptors and MEK because it was inhibited by ICI 118,551 (Samama et al., 1994) and by the selective MEK inhibitor U0126 (Favata et al., 1998) (Fig. 2A). ICI 118,551, which is an inverse agonist, did not inhibit basal ERK phosphorylation, suggesting that basal activity of the beta 2 receptor does not contribute significantly to the low level of phosphorylated ERK observed in unstimulated cultures.



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  		Figure 2.   Epinephrine-induced phosphorylation of ERK1 (gray bars) and ERK2 (black bars) is reduced by inhibitors of beta 2-adrenergic receptors and MEK and is independent of PKA and PKCepsilon . A, DRG cultures were treated with 1 µM epinephrine (Epi; n = 9) for 5 min in the absence or presence of (A) ICI 118,551 (ICI; 100 nM; n = 7) or U0126 (U; 10 µM; n = 2). B, Cultures were treated with 1 µM epinephrine (Epi; n = 11) for 5 min in the absence or presence of H89 (1 µM; n = 5) or calphostin C (Cal; 1 µM; n = 7). C, DRGs cultured from PKCepsilon wild-type (WT) or knock-out (KO) mice were treated with 1 µM epinephrine for 5 min (n = 3). Data are mean ± SE values. *p < 0.05 compared with phospho-ERK1 in epinephrine-treated cells; **p < 0.05 compared with phospho-ERK2 measured in epinephrine-treated cells (one-way ANOVA and Tukey's multiple comparison test).

Our previous studies indicated that signaling pathways involving PKA and PKCepsilon are important for primary afferent nociceptor sensitization induced by activation of beta 2-adrenergic receptors (Khasar et al., 1999a,b). Therefore, we examined whether these kinases lie in a signaling pathway that includes MEK and ERK in DRG neurons. We found that treatment with H89, which inhibits PKA (Chijiwa et al., 1990), or calphostin C, which inhibits several PKC isozymes including PKCepsilon (Mayne and Murray, 1998), did not reduce epinephrine-stimulated ERK phosphorylation (Fig. 2B). Moreover, treatment with epinephrine evoked similar levels of ERK phosphorylation in mouse DRG cultures obtained from wild-type and PKCepsilon null mice (Fig. 2C). These findings indicate that epinephrine stimulates ERK phosphorylation in DRG neurons through a signaling pathway that does not involve PKA or PKCepsilon .

We next evaluated whether MEK contributes to epinephrine-induced hyperalgesia and whether this is independent of PKA or PKCepsilon . Intradermal injection of epinephrine decreased mechanical nociceptive thresholds by ~35%, and this effect was inhibited by the MEK inhibitors U0126 and PD98059 (Favata et al., 1998) (Fig. 3A). However, U0126 and PD98059 had no effect on hyperalgesia induced by PGE2, which requires PKA activation for its pronociceptive effect (Aley and Levine, 1999; Chen and Levine, 1999), or by psi epsilon RACK, a specific activator of PKCepsilon (Dorn et al., 1999; Aley et al., 2000). Treatment with a dominant active MEK mutant was sufficient to induce hyperalgesia (Fig. 2D). This effect required the kinase activity of MEK because a kinase-dead MEK mutant was ineffective. Treatment with the specific PKCepsilon inhibitor epsilon V1-2 (Johnson et al., 1996; Khasar et al., 1999a) or with the PKA inhibitor WIPTIDE did not reduce hyperalgesia induced by active MEK (Fig. 2D). These findings indicate that MEK mediates epinephrine-induced mechanical hyperalgesia through a signaling pathway that is independent of PKA or PKCepsilon .



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  		Figure 3.   Epinephrine-induced hyperalgesia is mediated by MEK, independent of PKA and PKCepsilon . The mean ± SE baseline threshold before drug administration was 108.0 ± 0.5 gm (n = 162). A, Rats were treated intradermally with epinephrine (Epi; 100 ng; n = 12), UO126 (U; 1 µg; n = 6), UO126 plus epinephrine (U/Epi; n = 12), PD98059 (PD; 1 µg; n = 6), and PD98059 plus epinephrine (PD/Epi; n = 12). B, Rats were treated intradermally with PGE2 (100 ng; n = 6), UO126 plus PGE2 (U/PGE2; n = 6), and PD98059 plus PGE2 (PD/PGE2; n = 6). C, Rats were treated intradermally with the PKCepsilon agonist psi epsilon RACK (psi epsilon R; 1 µg; n = 6), UO126 plus PKCepsilon agonist (U/psi epsilon R; n = 6), and PD98059 plus PKCepsilon agonist (PD/psi epsilon R; n = 6). D, Rats were treated intradermally with active MEK (MEK+; 0.5 U; n = 12), inactive MEK (MEK-; 1 µg; n = 6), PKCepsilon inhibitor (epsilon V1-2; 1 µg; n = 6), PKCepsilon inhibitor plus active MEK (epsilon V1-2/MEK+; n = 6), WIPTIDE (WIP; 1 µg; n = 6), and WIPTIDE plus active MEK (WIP/MEK+; n = 6). *p < 0.05 by one-way ANOVA and Newman-Keuls test.

In human embryonic kidney 293 (HEK293) cells transfected to overexpress beta 2-adrenergic receptors, beta 2 agonists activate ERKs through a signaling cascade that involves a Gi/o-protein and Ras (Daaka et al., 1997; Della Rocca et al., 1997), whereas stimulation of endogenous beta 2 receptors activates ERKs through a pathway involving Gs and Rap-1 (Schmitt and Stork, 2000). To examine pathways involved in beta 2-mediated activation of ERKs in DRG neurons, we treated rat DRG cultures with pertussis toxin to inactivate Gi/o and with the isoprenylation inhibitor perillic acid (Hardcastle et al., 1999). Both of these agents reduced epinephrine-mediated mechanical hyperalgesia (Fig. 4A,B). Small GTPases are generally modified post-translationally by the addition of the isoprenaloids farnesyl or geranylgeranyl to a cysteine residue near the C terminus (Zhang and Casey, 1996). Ras proteins are preferentially farnesylated, and inhibitors of farnesyltransferase block the transforming ability of H-Ras (Kohl et al., 1994). In contrast, Rap1A and B, which have a leucine residue at their C termini, are preferentially geranylgeranylated. Treatment with FTase I, which inhibits Ras farnesylation, attenuated epinephrine-mediated mechanical hyperalgesia (Fig. 4C). Similarly, pertussis toxin and FTase I prevented epinephrine-induced phosphorylation of ERK1/2 (Fig. 5). These studies suggest that a Gi/o-Ras-ERK1/2 pathway contributes to epinephrine-induced hyperalgesia.



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  		Figure 4.   Epinephrine-induced mechanical hyperalgesia is mediated by a Gi/o-protein and Ras. The mean ± SE baseline threshold before drug administration was 108.0 ± 0.5 gm (n = 162). A, Rats were treated intradermally with epinephrine (Epi; 100 ng; n = 12), pertussis toxin (PTX; 1 µg; n = 6), or pertussis toxin plus epinephrine (PTX/Epi; n = 8). B, Rats were treated intradermally with epinephrine (Epi; 100 ng; n = 12), perillic acid (PER; 1 µg; n = 6), or perillic acid plus epinephrine (PER/Epi; n = 8). C, Rats were treated intradermally with epinephrine (Epi; 100 ng; n = 12), farnesyltransferase inhibitor I (FT; 1 µg; n = 6), or farnesyltransferase inhibitor I plus epinephrine (FT/Epi; n = 8). *p < 0.05 by one-way ANOVA and Newman-Keuls test.



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  		Figure 5.   Pertussis toxin (PTX) and farnesyltransferase inhibitor I (FT) inhibit epinephrine-induced ERK1/2 phosphorylation in DRG cultures. DRG cultures were treated with 100 nM pertussis toxin or 1 µM FTase I for 16 hr and then with or without 1 µM epinephrine (Epi) as indicated. Data are mean ± SE values from five to eight experiments. *p < 0.05 compared with phospho-ERK1 in epinephrine-treated cells; **p < 0.05 compared with phospho-ERK2 measured in epinephrine-treated cells (one-way ANOVA and Newman-Keuls test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Second-messenger signaling pathways involving PKA and PKCepsilon have been implicated previously in nociceptor sensitization (Khasar et al., 1999a,b). This report provides the first demonstration of a role for ERK signaling in this process. Using kinase-selective inhibitors, we found that epinephrine-induced phosphorylation of ERK1/2 is independent of PKA and PKCepsilon . In vivo, activated MEK was sufficient to cause a hyperalgesia that does not require PKA or PKCepsilon . Conversely, PKCepsilon - and PKA (PGE2)-mediated hyperalgesia was independent of MEK activity. Therefore, ERKs, PKA, and PKCepsilon appear to define three independent signaling pathways that mediate nociceptor sensitization by inflammatory mediators.

beta 2-adrenergic receptor activation stimulates ERK phosphorylation in HEK293 cells (Daaka et al., 1997; Della Rocca et al., 1997; Schmitt and Stork, 2000), COS-7 cells (Maudsley et al., 2000), S49 lymphoma cells (Wan and Huang, 1998), and cardiac myocytes (Zou et al., 1999). Some of the most detailed studies have been performed with HEK293 cells in which endogenous beta 2 receptors activate ERKs through a pathway involving Gs, PKA, Rap1, and B-Raf (Schmitt and Stork, 2000). It is unlikely that Rap1 plays a role in beta 2 receptor-mediated nociceptor sensitization because treatment with the PKA inhibitor H89 did not block epinephrine-induced ERK phosphorylation or mechanical hyperalgesia. In HEK293 cells transfected to overexpress beta 2 receptors, epinephrine stimulates a different pathway resulting in activation of Gi/o and Src, and transactivation of EGF receptors leading to stimulation of Ras, MEK, and ERKs (Daaka et al., 1997; Della Rocca et al., 1997; Maudsley et al., 2000). We found evidence to support involvement of Gi/o and Ras in beta 2 receptor-mediated ERK activation in DRG neurons and hyperalgesia.

In addition to regulating gene expression, cell proliferation, differentiation, development, and apoptosis (Lewis et al., 1998), ERKs have been implicated in neural plasticity associated with learning and memory (Bailey et al., 1997; Martin et al., 1997). Here we demonstrate a role for ERK signaling in another form of neural plasticity, namely sensitization of nociceptors. The downstream effectors of ERKs that mediate nociceptor sensitization are not known. Because modulation of voltage-sensitive potassium channels may contribute to nociceptor sensitization (Evans et al., 1999), recent findings that the A-type K+ channel Kv4.2 is a substrate for ERK1/2 (Adams et al., 2000) may provide such a downstream target. Production of arachidonic acid metabolites by cytoplasmic phospholipase A2, another substrate of ERK1/2 (Lin et al., 1993), has been implicated in signaling pathways involved in nociceptor function (Hwang et al., 2000; Piomelli, 2001). Thus, at least two known ERK1/2 substrates are potential mediators of nociceptor sensitization induced by ERK signaling.

Activation of ERKs by beta -adrenergic receptor stimulation may contribute to inflammatory pain, because increased levels of epinephrine are found at sites of inflammation (Mikhailov and Rusanova, 1993) and beta -adrenergic receptor antagonists reduce inflammatory hyperalgesia (Cunha et al., 1991). Catecholamines released from sympathetic nerve terminals and from the adrenal medulla also appear to contribute to sympathetically maintained pain and stress-aggravated pain (Choi and Rowbotham, 1997; Khasar et al., 1998b). When injected intradermally, epinephrine lowers the nociceptive threshold with an ED50 of ~20 ng (Khasar et al., 1999b), which is similar to the ED50 for bradykinin and PGE2 (Khasar et al., 1993). In addition to catecholamines, NGF (Safieh-Garabedian et al., 1997) and bradykinin (Levine and Taiwo, 1994) are two other hyperalgesic mediators released during inflammation and tissue damage that can activate ERK1/2 (Clark and Murray, 1995; Hundle et al., 1995). This suggests that ERK signaling plays an important role in pain states evoked by several different mediators and pathological conditions. ERK pathways have several components, affording an opportunity for antagonism at many levels. Therefore, inhibition of ERK signaling in nociceptors may provide a fruitful strategy for the discovery of novel analgesics.


    FOOTNOTES

Received April 6, 2001; revised June 11, 2001; accepted June 15, 2001.

This work was supported by Public Health Service Grants NS21647 (J.D.L.) and AA10036 (R.O.M.).

K.O.A. and A.M. contributed equally to this work.

Correspondence should be addressed to Dr. Robert O. Messing, Ernest Gallo Clinic and Research Center at the University of California, San Francisco, 5858 Horton Street, Suite 200, Emeryville, CA 94608. E-mail: romes{at}itsa.ucsf.edu.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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