Nociceptor sensitization by extracellular signal-regulated kinases

Abstract

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 G(i/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.

Fig. 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.

Fig. 2.

Epinephrine-induced phosphorylation of ERK1 (gray bars) and ERK2 (black bars) is reduced by inhibitors of β₂-adrenergic receptors and MEK and is independent of PKA and PKCε. 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 μmepinephrine (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 PKCε wild-type (WT) or knock-out (KO) mice were treated with 1 μmepinephrine 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).

Fig. 3.

Epinephrine-induced hyperalgesia is mediated by MEK, independent of PKA and PKCε. 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 PKCε agonist ψεRACK (ψεR; 1 μg;n = 6), UO126 plus PKCε agonist (U/ψεR; n = 6), and PD98059 plus PKCε agonist (PD/ψεR; n = 6).D, Rats were treated intradermally with active MEK (MEK+; 0.5 U; n = 12), inactive MEK (MEK−; 1 μg; n = 6), PKCε inhibitor (εV1–2; 1 μg; n = 6), PKCε inhibitor plus active MEK (εV1–2/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.

Fig. 4.

Epinephrine-induced mechanical hyperalgesia is mediated by a G_i/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.

Fig. 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).