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, 24 (38), 8310-21

Ionotropic and Metabotropic Receptors, Protein Kinase A, Protein Kinase C, and Src Contribute to C-fiber-induced ERK Activation and cAMP Response Element-Binding Protein Phosphorylation in Dorsal Horn Neurons, Leading to Central Sensitization

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Ionotropic and Metabotropic Receptors, Protein Kinase A, Protein Kinase C, and Src Contribute to C-fiber-induced ERK Activation and cAMP Response Element-Binding Protein Phosphorylation in Dorsal Horn Neurons, Leading to Central Sensitization

Yasuhiko Kawasaki et al. J Neurosci.

Abstract

Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons and its contribution to central sensitization have been investigated. In adult rat spinal slice preparations, activation of C-fiber primary afferents by a brief exposure of capsaicin produces an eightfold to 10-fold increase in ERK phosphorylation (pERK) in superficial dorsal horn neurons. The pERK induction is reduced by blockade of NMDA, AMPA/kainate, group I metabotropic glutamate receptor, neurokinin-1, and tyrosine receptor kinase receptors. The ERK activation produced by capsaicin is totally suppressed by inhibition of either protein kinase A (PKA) or PKC. PKA or PKC activators either alone or more effectively together induce pERK in superficial dorsal horn neurons. Inhibition of calcium calmodulin-dependent kinase (CaMK) has no effect, but pERK is reduced by inhibition of the tyrosine kinase Src. The induction of cAMP response element binding protein phosphorylation (pCREB) in spinal cord slices in response to C-fiber stimulation is suppressed by preventing ERK activation with the MAP kinase kinase inhibitor 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) and by PKA, PKC, and CaMK inhibitors. Similar signaling contributes to pERK induction after electrical stimulation of dorsal root C-fibers. Intraplantar injection of capsaicin in an intact animal increases expression of pCREB, c-Fos, and prodynorphin in the superficial dorsal horn, changes that are prevented by intrathecal injection of PD98059. Intrathecal PD98059 also attenuates capsaicin-induced secondary mechanical allodynia, a pain behavior reflecting hypersensitivity of dorsal horn neurons (central sensitization). We postulate that activation of ionotropic and metabotropic receptors by C-fiber nociceptor afferents activates ERK via both PKA and PKC, and that this contributes to central sensitization through post-translational and CREB-mediated transcriptional regulation in dorsal horn neurons.

Figures

Figure 3.
Figure 3.
ag, Involvement of ERK, PKA, PKC, and CaMK in C-fiber stimulation-induced pCREB in the superficial dorsal horn of spinal cord slices. ac, C-fiber electrical stimulation (Elec stim) induces pCREB in the medial superficial dorsal horn. c is a high magnification of the area in b indicated by the square. d, Inhibition of ERK activation with the MEK inhibitor PD98059 (50 μm) blocks the C-fiber-induced pCREB increase. PD98059 was incubated for 30 min before the electrical stimulation. The dotted lines indicate the top border of the gray matter of the superficial dorsal horn. Scale bars, 100 μm. e, Numbers of pCREB-IR nuclei in laminas I-II in the medial two thirds of the dorsal horn per 15-μm-thick section. **p < 0.01, compared with control; ++p < 0.01, compared with electrical stimulation without PD98059 (n = 4). PD98059 had no effect on basal pCREB levels. The spinal slices were fixed 5 min after electrical stimulation for pCREB immunostaining. f, Time course of capsaicin (3 μm; 5 min) induced pERK and pCREB increases in the superficial dorsal horn. At all of the time points, pERK (F(4, 15) = 16.982; p < 0.0001) and pCREB (F(4, 13) = 3.837; p = 0.0028) levels are significantly higher than control levels (p < 0.05; n = 3–5). g, Numbers of pCREB-IR neurons in the superficial dorsal horn (laminas I-II) after capsaicin stimulation (3 μm; 5 min) in the presence of MEK inhibitor PD98059 (PD), PKA inhibitor H89, PKC inhibitor Ro-31–84-25 (Ro), and CaMK inhibitor KN93. ANOVA indicates an overall effect of these inhibitors on pCREB expression (F(6,25) = 10.592; p < 0.0001). ++ p < 0.01, compared with control; *p < 0.05, **p < 0.01, compared with capsaicin (n = 4–6). Six to eight sections (15 μm) were analyzed for each slice. The slices were incubated with PD98059 for 30 min and the rest of the drugs for 10 min and fixed 10 min after the capsaicin stimulation.
Figure 1.
Figure 1.
ad, pERK immunostaining in the dorsal horn of intact rats after intrathecal injection of saline (a), NMDA (0.5 μg; b), substance P (SP) (10 μg; c), and BDNF (5 μg; d). Animals were perfused 10 min after the injection. Scale bars, 100 μm. e, Numbers of pERK-IR neurons per 15-μm-thick section in the superficial (laminas I-II) dorsal horn. ANOVA indicates an overall effect of these agents on pERK expression (F(3,12)=57.595; p<0.0001). *p<0.01, compared with control (n = 4).
Figure 2.
Figure 2.
ac, pERK immunofluorescence in control unstimulated (a), KCl depolarized (b), and capsaicin-stimulated (c) spinal cord slices. The slices were fixed 10 min after the capsaicin exposure (3μm; 5 min) and KCl (90 mm; 5 min) depolarization. The dotted lines indicate the top border of the gray matter of the spinal cord. d, e, pERK immunostaining by DAB in control unstimulated and electrical C-fiber stimulation (1 mA, 50 Hz; 50 msec; 150 pulses) of an attached dorsal root in spinal cord slices. The slices were fixed 2 min after electrical stimulation. Scale bars, 100μm. f, Numbers of pERK-IR neurons after exposure of spinal cord slices to NMDA (100 μm), substance P (SP; 100 μm), and BDNF (200 ng/ml). ANOVA indicates an overall effect of these agents on pERK expression (F(3, 11) = 18.489; p < 0.0001). *p < 0.01, compared with control (n = 4–5). All of the slices were fixed 10 min after exposure.
Figure 4.
Figure 4.
a, b, Involvement of ionotropic, metabotropic, and tyrosine kinase receptors in capsaicin-induced pERK induction in spinal cord slices. a, Number of pERK-IR neurons in the superficial dorsal horn (laminas I-II) after capsaicin stimulation (3 μm; 5 min) in the presence of the NMDA receptor antagonist MK-801 (50 and 100 μm), the AMPA receptor antagonist CNQX (10 and 20μm), and mGluR1 antagonist CPCCOEt (CP; 1 and 10μm). ANOVA indicates an overall effect of these agents on pERK expression (F(7, 33) = 29.782; p < 0.0001). Mean ± SEM; *p < 0.05; **p < 0.01, compared with capsaicin (n = 4–6). b, Number of capsaicin-induced pERK-IR neurons in the presence of the NK-1 receptor antagonist GR205171A (GR; 50 and 100 μm) and the Trk inhibitor K252a (50 and 100 nm). ANOVA indicates an overall effect of these agents on pERK expression (F(5, 25) = 16.442; p < 0.0001). *p < 0.05; **p < 0.01, compared with capsaicin (n = 4–6). Six to eight sections (15μm) were included for each slice. The slices were incubated with each drug for 5–10 min and fixed 10 min after the capsaicin stimulation.
Figure 5.
Figure 5.
ad, pERK immunofluorescence in control unstimulated (a) and after exposure to the PKA activator forskolin (20 μm)(b), the PKC activator PMA (5 μm)(c), and forskolin plus PMA (d). Scale bar, 100μm. The dotted lines indicate the top border of the gray matter of the spinal cord. e, Numbers of pERK-IR neurons in the superficial dorsal horn after each treatment. ANOVA indicates an overall effect of these agents on pERK expression (F(6, 22)=16.442;p<0.0001).*p<0.05;**p< 0.01 (n = 3–5). All of the slices were fixed 20 min after drug exposure.
Figure 6.
Figure 6.
a, b, Involvement of PKA, PKC, Src, and CaMK in capsaicin-induced pERK induction in spinal cord slices. a, Numbers of pERK-IR neurons in the superficial dorsal horn (laminas I-II) after capsaicin stimulation (3 μm; 5 min) in the presence of the PKA inhibitor H89 (0.1 and 1 μm), the PKC inhibitor Ro-31–84-25 (Ro; 0.1 and 1 μm), and H89 plus Ro-31–84-25 (0.1 μm). ANOVA indicates an overall effect of these inhibitors on pERK expression (F(6, 27) = 14.438; p < 0.0001). **p < 0.01, compared with capsaicin. Mean ± SEM (n = 3–6). b, Numbers of capsaicin-induced pERK-IR neurons in the presence of the general tyrosine kinase(TK) inhibitor genistein (50 and 100μm), the Srcinhibitor PP2 (5 and 20μm), and the CaMK inhibitor KN93 (20 μm).ANOVA indicates an overall effect of these agents on pERK expression (F(7, 23) = 4.327; p = 0.003). *p < 0.05; **p < 0.01, compared with capsaicin (n=3–6).Six to eight sections (15 μm) were analyzed for each slice. Mean ± SEM. The slices were incubated with each drug for 10 min and fixed 10 min after the capsaicin stimulation.
Figure 7.
Figure 7.
ah, ERK activation contributes to the capsaicin-induced phosphorylation of CREB and the expression of c-fos and prodynorphin in vivo. ad, Intraplantar injection of capsaicin induces pERK (a, c) and pCREB (b, d) in the ipsilateral dorsal horn. ad were obtained from the same spinal sections. e, pERK is largely colocalized with pCREB in the nuclei of individual neurons in the medial laminas I and II of the dorsal horn. e is a high magnification of the areas in c and d indicated by the square. Animals were perfused 5 min after capsaicin injection. Scale bars, 100μm. f, Intraplantar capsaicin induces pERK and c-Fos in nuclei of neurons in medial laminas I and II. The arrows indicate double-stained neurons. Scale bar, 30 μm. g, Intraplantar capsaicin induces an increase in prodynorphin-IR neurons in the medial dorsal horn 3 hr after capsaicin injection, which is suppressed by intrathecal PD98059 (1μg) administered 30 min before capsaicin injection. Control animals received intrathecal vehicle (10% DMSO) and an intraplantar injection of saline with 10% Tween 80. Scale bar, 100μm. h, RNase protection assay showing a transient increase of prodynorphin mRNA levels in the ipsilateral dorsal horn after intraplantar capsaicin. Actin is used as the loading control. The fold change for the density of prodynorphin mRNA bands is calculated after normalization with actin.
Figure 8.
Figure 8.
ERK activation is involved in mediating capsaicin-induced secondary mechanical allodynia. Capsaicin injection (20 μg in 20 μl) into the heel of a rat hindpaw induces a secondary mechanical allodynia in the center of the plantar surface of the hindpaw, which is inhibited by intrathecal injection of the MEK inhibitor PD 98059 (1 μg) 30 min before capsaicin. **p < 0.01, compared with vehicle control (10% DMSO); t test; n = 7. Data are expressed as percentage of precapsaicin control. The MEK inhibitor had no effect on basal pain sensitivity. Mechanical allodynia was measured with von Frey filaments.

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