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. 2005 Jun 29;25(26):6119-26.
doi: 10.1523/JNEUROSCI.0285-05.2005.

Epac mediates a cAMP-to-PKC signaling in inflammatory pain: an isolectin B4(+) neuron-specific mechanism

Tim B Hucho  1 , Olayinka A Dina, Jon D Levine
Affiliations

Epac mediates a cAMP-to-PKC signaling in inflammatory pain: an isolectin B4(+) neuron-specific mechanism

Tim B Hucho et al. J Neurosci. .

Abstract

The epsilon isoform of protein kinase C (PKCepsilon) has emerged as a critical second messenger in sensitization toward mechanical stimulation in models of neuropathic (diabetes, alcoholism, and cancer therapy) as well as acute and chronic inflammatory pain. Signaling pathways leading to activation of PKCepsilon remain unknown. Recent results indicate signaling from cAMP to PKC. A mechanism connecting cAMP and PKC, two ubiquitous, commonly considered separate pathways, remains elusive. We found that, in cultured DRG neurons, signaling from cAMP to PKCepsilon is not mediated by PKA but by the recently identified cAMP-activated guanine exchange factor Epac. Epac, in turn, was upstream of phospholipase C (PLC) and PLD, both of which were necessary for translocation and activation of PKCepsilon. This signaling pathway was specific to isolectin B4-positive [IB4(+)] nociceptors. Also, in a behavioral model, cAMP produced mechanical hyperalgesia (tenderness) through Epac, PLC/PLD, and PKCepsilon. By delineating this signaling pathway, we provide a mechanism for cAMP-to-PKC signaling, give proof of principle that the mitogen-activated protein kinase pathway-activating protein Epac also stimulates PKC, describe the first physiological function unique for the IB4(+) subpopulation of sensory neurons, and find proof of principle that G-protein-coupled receptors can activate PKC not only through the G-proteins alpha(q) and betagamma but also through alpha(s).

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Figures

Figure 1.
Figure 1.
β2-AR agonist induced translocation of PKCϵ in cultured DRG neurons. A, Confocal images of representative untreated (left) versus isoproterenol-treated (1 μm, 30 s; right) cultured DRG neurons. Cultures were fixed after treatment, incubated with the affinity-purified PKCϵ-specific antiserum SN134 (1:1000), and detected with FITC-coupled donkey anti-rabbit IgG serum (1:200). Insets, Enlarged area, indicated by the white frame. After isoproterenol treatment, a portion of PKCϵ can be seen to translocate to the plasma membrane. Scale bar, 20 μm. B, Percentage of neurons demonstrating, after 30 s, PKCϵ translocation to the plasma membrane in response to indicated concentrations of isoproterenol. C, Percentage of neurons demonstrating PKCϵ translocation with isoproterenol (1 μm) stimulation, for indicated time points. Filled bars represent cultures treated with 1 μm isoproterenol. Cultures represented by dotted bars were pretreated for 15 min with the β2-AR-specific inhibitor ICI 118,551 (50 μm) before stimulation with 1 μm isoproterenol. **p < 0.01. Error bars represent SEM.
Figure 2.
Figure 2.
G-protein αs, adenylyl cyclase, and Epac, but not PKA, are involved in PKCϵ translocation. A, DRG cultures were pretreated for 15 min with indicated concentrations [1, 10, 100, and 1000× the CMIQ-IC50 (0.03-30 μm)] of the PKA-specific inhibitor CMIQ (Lu et al., 1996) before stimulation with 1 μm isoproterenol for 30 s. Cultures not treated with either CMIQ or isoproterenol served as negative controls. B, Injection of the PKA inhibitor CMIQ (2.5 μg/2.5 μl) intradermally in rat paws did not change the mechanical paw-withdrawal threshold. Injection of active PKA [PKA catalytic subunit (PKAcs); 25 U/2.5 μl] induced robust mechanical hyperalgesia. Preinjection of CMIQ 15 min before the injection of PKAcs completely blocked the PKA-induced hyperalgesia in vivo. C, Cultures were stimulated with activators of G-protein αs (cholera toxin; 1 μg/ml), adenylyl cyclase (forskolin, 5 μm), and Epac (CPTOMe, 10 μm), for the indicated time. Unstimulated cells served as a negative control, and isoproterenol (1 μm, 30 s)-treated cells served as a positive control. *p < 0.05; **p < 0.01. Error bars represent SEM.
Figure 3.
Figure 3.
β2-AR-induced Epac-mediated translocation of PKCϵ requires the activity of both PI-PLC and PLD. A, Percentage of neurons demonstrating PKCϵ translocation to the plasma membrane after stimulation with isoproterenol (Iso; 1 μm, 30 s). Cultures were pretreated for 15 min with the indicated inhibitors: PC-PLC inhibitor (D-609; 30 μm), PI-PLC inhibitor (U73122; 10 μm), inactive homolog of U73122 (U73343; 10 μm), PLD inhibitor (1-butanol; 50 mm), inactive homolog for 1-butanol (2-butanol; 50 mm); PKC kinase inhibitor (BIM; 100 nm). Concentrations used are ∼10 times the reported IC50 values. B, Percentage of neurons demonstrating PKCϵ translocation to the plasma membrane after stimulation with the Epac-specific activator CPTOMe (10 μm, 90 s). Inhibitors were used as in A. **p < 0.01. Error bars represent SEM.
Figure 4.
Figure 4.
A-D, PKCϵ translocation and IB4 double-staining epifluorescence images of double-stained cells tested for PKCϵ (A and C; 1:1000 diluted) and IB4 (B and D; 1:100 diluted). A, C, Although the cell in the top row is translocating PKCϵ to the plasma membrane (A), it also shows clear plasma membrane staining of the IB4 epitope (C). In contrast, the cell in C does not translocate PKCϵ and is not positive for the IB4 epitope (D). Insets, Enlarged area indicated by the white frame. Scale bar, 20 μm.
Figure 5.
Figure 5.
In vivo Epac mediates epinephrine-induced hyperalgesia via PI-PLC, PLD, and PKCϵ. A, Injection of epinephrine (0.1 μg in 2.5 μl) and CTPOMe (6.3 μg in 2.5 μl) produce hyperalgesia of similar magnitude, whereas saline vehicle injection has no effect. CPTOMe-induced sensitization can be completely blocked by the preinjection of the specific PKCϵ inhibitory peptideϵV1-2 (1 μg in 2.5 μl) 30 min before stimulation with CPTOMe, demonstrating that, in vivo, Epac also induces mechanical hyperalgesia through activation of PKCϵ. B, Epinephrine-induced (filled bars) and CPTOMe-induced (dotted bars) mechanical hyperalgesia can be completely blocked by preinjection of the PI-PLC inhibitor U73122 (2.5 μl, 1 μg/μl), but not its inactive control, U73343 (2.5 μl, 1 μg/μl), 30 min before epinephrine/CPTOMe stimulation. Likewise, resembling our in vitro data, the injection of the PLD inhibitor 1-butanol (2.5 μl, 10.9 m), but not its inactive control, 2-butanol, completely inhibits the sensitization through epinephrine/CPTOMe injection. The inhibitors show little or no effect on saline control injections (open bars). Both phospholipases are therefore also necessary for the mediation of β2-AR-stimulated/Epac/PKCϵ-mediated mechanical hyperalgesia in vivo. **p < 0.01. Error bars represent SEM.
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Comment in

  • An Epac-dependent pain pathway.
    Griffin RS. Griffin RS. J Neurosci. 2005 Sep 7;25(36):8113-4. doi: 10.1523/JNEUROSCI.3044-05.2005. J Neurosci. 2005. PMID: 16148218 Free PMC article. Review. No abstract available.

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