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Biological

Time-dependent Activation of ERK1/2 in Nerve Terminals of the Dentin-Pulp Complex following Bradykinin Treatment

Y. Korkmaz1,*, W. Bloch2, K. Schneider1, S. Zimmer3, K. Addicks4 and W.H.-M. Raab1

1 Dept. of Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany;
2 Dept. of Molecular and Cellular Sports Medicine, German Sports University, Cologne, Germany;
3 Dept. of Operative Dentistry, University Witten/Herdecke, Witten, Germany; and
4 Dept. I of Anatomy, University of Cologne, Germany

Correspondence: * corresponding author, yueksel.korkmaz{at}uni-duesseldorf.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors, and the inflammatory mediator bradykinin (BK) led to a reduced threshold in the nociceptor terminals, activating intracellular signaling by phosphorylating receptors and ion channels. The effects of BK on the non-transcriptional modulation of the ERK1/2 in the peripheral nociceptor terminals, including in nerve endings of the dentin-pulp complex, are unknown. The time-dependent effects of BK (10–7 M) on the ERK1/2 phosphorylation in nerve terminals of the dentin-pulp complex were investigated by quantitative and double immunolabeling with organ bath experiments. In nerve terminals, total and p-ERK1/2 were detected. In comparison with the controls, the numbers of p-ERK1/2-positive nerve endings increased after 1 and 3 min and decreased after 10 min of BK treatment. Analysis of the data indicates that BK induces phosphorylation-mediated local activation of ERK1/2 in nerve terminals modulating nociception in the dentin-pulp complex.

Key Words: mitogen-activated protein kinase (MAPK) • extracellular signal-regulated protein kinase 1/2 (ERK1/2) • bradykinin • peripheral sensitization • dentin nociception


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptors (Julius and Basbaum, 2001). In addition to the inflammatory mediators, such as prostaglandin E2 (PGE2) and nerve growth factor (NGF), the mediator bradykinin (BK) is released during inflammation (Julius and Basbaum, 2001). The inflammatory mediators, including BK, act on G-protein-coupled receptors, expressed on nociceptors, and activate intracellular signaling by phosphorylating receptors and ion channels in the nociceptor terminals (Julius and Basbaum, 2001). BK induces changes in the sensory-neuron-specific voltage-gated Na+ channel Nav1.8, altering its activation (Julius and Basbaum, 2001; Ji et al., 2003). These changes increase the sensitivity and excitability of the nociceptor terminal and are referred to as ‘peripheral sensitization’ (Julius and Basbaum, 2001; Ji et al., 2003).

Binding of ligands to the tyrosine kinase receptors or G-protein-coupled receptors leads to the activation of the GTPase Ras, which recruits the mitogen-activated protein kinase kinase kinase (MAPKKK) Raf to the cell membrane. Activated Raf phosphorylates and activates the MAP kinase kinases (MAPKK) MEK1/MEK2, which in turn activate the effector MAP kinases (MAPKs), the extracellular signal-regulated kinases 1 and 2 (ERK1/2), by phosphorylation of the specific threonine (Thr) and tyrosine (Tyr) residues. MAPKs require dual phosphorylation in 2 neighboring Thr and Tyr residues within their activation loop for full activation (Thomas and Huganir, 2004).

MAPKs are involved in pain mediation by different factors and inflammatory mediators (Ji and Woolf, 2001; Ji et al., 2002; Obata et al., 2003; Ji, 2004). In addition to the regulation of gene expression, cell proliferation, and cell differentiation, ERK1/2 have been implicated in the inflammation-dependent sensitization of nociceptors (Fields et al., 1997; Ji et al., 1999; Aley et al., 2001; Ji and Woolf, 2001). The activation of ERK1/2 in dorsal horn neurons is involved in the generation of central sensitization, and ERK1/2 activation in peripheral terminals of the primary sensory neurons contributes to peripheral sensitization (Aley et al., 2001; Dai et al., 2002). In addition to transcriptional or translational regulation, post-translational regulation also contributes to peripheral sensitization (Ji et al., 2003).

It is important to understand the localization and the function of the neurotransmitters, receptors, and transducers that are expressed by nociceptors of the dentin-pulp complex, because the neurotransmission mechanisms of nociception in the dentin-pulp complex are not fully understood (Byers, 1984). In the isolated terminals of the dental pulp, BK evokes neurosecretion via a BK receptor 2 (B2)-dependent mechanism (Goodis et al., 2000). However, the post-translational ERK1/2 activation in the nerve terminals of the dentin-pulp-complex by their response to inflammatory mediator BK is unknown. To test whether there is a non-transcriptional constitutive activity of ERK1/2 in peripheral nerve fibers, we examined the localization of the ERK1/2 in nerve fibers of the rat molar dentin-pulp complex using total and phospho-specific antibodies to the ERK1/2. To test the stimulation of BK upon non-transcriptional activation of ERK1/2 in pulpal nerve fibers, we performed organ bath experiments.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Statement on Animal Use and Care
Male Wistar rats (n = 12; 3 mos old, weighing from 250 to 280 g each) were housed at 22°C, on a standard light/dark schedule with access to food and water ad libitum. Animal handling procedures were carried out in compliance with the guidelines of the Heinrich-Heine-University Düsseldorf.

Tissue Preparation and Bradykinin Treatment
To examine the localization and constitutive activation of ERK1/2 in nerve fibers of the dentin-pulp complex, we anesthetized the rats with a mixture of Ketamine (100 mg/kg) and Xylazine (5 mg/ kg) and transcardially perfused them with a fixative containing 4% paraformaldehyde (PFA) + 0.2% picric acid in 0.1 M phosphate-buffered saline (PBS), pH 7.4, for 24 hrs (Appendix 1).

To test the reaction to stimulation with BK on phosphorylation of ERK1/2 in nerve fibers of the dentin-pulp complex, we performed organ bath experiments (Korkmaz et al., 2006). Male Wistar rats (n = 12) were killed by exposure, by inhalation, to an increasing concentration of CO2. Three molars of one half of the right jaws (maxillary/mandibular) were dissected free from the roots by means of forceps and placed in Tyrode solution (pH 7.4) with the following composition (in mM): CaCl2 x 2H2O, 1.8; MgCl2 x 6H2O, 1.1; KCl, 5.4; NaCl, 136.9; NaH2PO4, 0.4; glucose, 10.1; and NaHCO3, 23.8 (Merck, Darmstadt, Germany). The molars were treated with 10 mL Tyrode solution containing 10–7 M BK (Sigma, St. Louis, MO, USA) for 1, 3, and 10 min at 37°C (Appendix 2). The control molars of left jaws (mandibular/maxillary) were treated only with Tyrode solution for 1, 3, and 10 min without BK (Appendix 3).

Immunohistochemistry
Reagents and Antibodies
The rabbit anti-t-ERK1/2 polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). The monoclonal anti-p-ERK1/2 antibody (di-phosphorylated ERK1/2, mouse IgG1 isotype) (Yung et al., 1997) was obtained from Sigma. The activation of ERK was determined by the detection of Thr183 and Tyr185 dual phosphorylation of ERK1/2 (Appendix 4).

Avidin-Biotin-Peroxidase Complex Method
In the free-floating sections, endogenous peroxidases were inhibited with 0.3% H2O2. To block non-specific bindings, we treated sections with 1% bovine serum albumin (BSA) + 10% normal goat serum (NGS). Thereafter, sections were incubated for 48 hrs at 4°C with anti-ERK1/2 (1:1000) and anti-diphospho-ERK1/2 (1:1000), followed by incubation with biotin-conjugated goat anti-rabbit IgG (1:500) and biotinylated anti-mouse IgG (1:500), respectively. The sections were then treated with avidin-biotin-peroxidase complex (1:100) for 1 hr. The reaction was visualized with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.01% H2O2 and 0.01% nickel ammonium sulphate (Appendix 5).

Double Immunofluorescence Labeling
The free-floating sections were incubated with 10% NGS + 2% BSA for 30 min. Then, sections were incubated with mouse anti-p-ERK1/2 (1:500) overnight at 4°C. The sections were incubated with DyLightTM 549-conjugated goat anti-mouse IgG (1:1000) for 1 hr at RT and with the rabbit anti-PGP 9.5 (1:800) for 1 hr at 4°C in the dark. Thereafter, the sections were incubated with biotinylated goat anti-rabbit IgG (1:1000) for 1 hr and with the DyLightTM 488-conjugated NeutrAvidinTM (1:200) for 1 hr at RT in the dark (Appendix 6).

Image Acquisition and Quantification of Nerve Fibers
The number of t-ERK1/2-, p-ERK1/2-positive nerve fibers in the dentin-pulp complex was counted by the point-counting method. PGP 9.5-stained nerve fibers were quantified for the positive control of p-ERK1/2 in nerve fibers (Appendix 7).

Confocal Analysis of Double Staining
Analyses of the double-labeling and images were acquired by means of an LSM510 confocal microscope (Zeiss, Oberkochen, Germany) (Klinz et al., 2007).

Statistical Analysis
Statistical differences in nerve fiber number between control and BK treatment were analyzed by the two-tailed Student’s t test. One-way ANOVA with the Bonferroni post hoc test was used to compare multiple means. Data are presented as means ± SD. Statistical significance was considered at a p value ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of t-ERK1/2 and p-ERK1/2 in Nerve Terminals of the Dentin-Pulp Complex under Basal Conditions
In the root (Fig. 1AGo) and coronal pulp (Figs. 1B–1DGo), t-ERK1/2 was detected in nerve fibers. The t-ERK1/2 was identified in all odontoblasts, with different staining intensities (Figs. 1A–1DGo). Nerve fibers showing immunoreactivity to t-ERK1/2 were detected beneath (Fig. 1CGo) and in the odontoblast layer (Fig. 1DGo).


Figure 1
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  		Figure 1. The constitutive localization of t-ERK1/2 and p-ERK1/2 in nerve fibers of the dentin-pulp complex. Nerve fibers of the root pulp showed immunoreactivity for t-ERK1/2 (A). In the coronal pulp, t-ERK1/2-positive nerve fibers were detected as free fibers and in association with blood vessels (B). Odontoblasts were strongly stained by t-ERK1/2 (B; double arrows). Nerve fibers beneath the odontoblast layer (C; double arrows) in the odontoblast layer (D; double arrows) revealed a localization of the t-ERK1/2. In thick nerve fibers accompanying the blood vessels (asterisks) in the root pulp, p-ERK1/2 was identified (e). In the coronal pulp, p-ERK1/2-positive nerve fibers were distributed in the subodontoblastic plexus as perivascular and free nerve fibers (F). In a subpopulation of odontoblasts, p-ERK1/2 was detected (F, G; arrows). The p-ERK1/2 immunoreactive nerve fibers were distributed in the coronal pulp (F, G) and penetrated the odontoblast layer (H). In the predentin, p-ERK1/2-labeled nerve endings were detected (H; double arrows). Some odontoblast processes revealed p-ERK1/2 immunoreactivity (H; arrows). Bar: A-G = 120 µm, H = 60 µm; rp = root pulp, cp = coronal pulp, o = odontoblasts, pd = predentin, d = dentin.

 
The p-ERK1/2-positive nerve fibers pass through the radicular pulp (Fig. 1EGo) with little branching, but form numerous branches in the coronal pulp (Fig. 1FGo). The localization of p-ERK1/2 was detected in the perivascular as well as in free nerve fibers of the dental pulp (Figs. 1E–1HGo). In a subpopulation of odontoblasts, p-ERK1/2 was identified (Figs. 1F–1HGo). The p-ERK1/2 immunoreactive nerve fibers were distributed in the subodontoblastic plexus, reached the predentin, and penetrated initial dentinal tubules (Fig. 1HGo).

The immunohistochemical controls resulted in the disappearance of the signal product (data not shown).

Effects of BK on t-ERK1/2 and PGP 9.5 in Nerve Terminals of the Dentin-Pulp Complex after 1, 3, and 10 min of BK Treatment
t-ERK1/2 was detected without significant differences in nerve fibers and nerve-ending numbers of control tissues and after BK treatment for 1 (Figs. 2A, 2BGo), 3 (Figs. 2C, 2DGo), and 10 (Figs. 2E, 2FGo) min (Fig. 2GGo). The immunohistochemical signals were absent in the consecutive sections of the BK-control and BK-treatment conditions when the secondary and primary antibodies were omitted in separate incubations (Appendix Fig. 1).


Figure 2
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  		Figure 2. Effects of BK on t-ERK1/2 in nerve fibers and nerve endings of the dentin-pulp complex after 1, 3, and 10 min of BK treatment. The t-ERK1/2-labeled nerve fibers were detected without significant differences in their numbers for one-minute BK-control (A; G = 0.1210 ± 0.0136) and after one-minute BK treatment (B; G = 0.1173 ± 0.0293). In the subodontoblastic plexus, t-ERK1/2 was detected in nerve fibers (B; double arrows). The number of t-ERK1/2 immunoreactive nerve fibers revealed no significant change in control (C; G = 0.1303 ± 0.0188) and after three-minute BK stimulation (D; G = 0.1170 ± 0.0029). In the odontoblast layer, t-ERK1/2 was identified in nerve varicosities (E; double arrows). The changes in the numbers of t-ERK1/2 immunoreactive nerve fibers and nerve endings in BK-control (e; G = 0.1095 ± 0.0247) and after BK stimulation (F; G = 0.1285 ± 0.0137) were not significant. Bar: A-F = 120 µm; rp = root pulp, cp = coronal pulp, o = odontoblasts, pd = predentin, d = dentin. The histogram is defined as G.

 
In the dentin-pulp complex, there were no significant differences in PGP 9.5-positive nerve fibers and nerve-ending numbers between BK-untreated and BK-treated for 1, 3, and 10 min (Appendix Fig. 2).

The Co-localization of p-ERK1/2 and PGP 9.5 in Nerve Terminals of the Dentin-Pulp Complex
DRAQ5, a chromatin marker, was detected in cell nuclei of the dental pulp (Figs. 3A, 3EGo). Under BK-control (Figs. 3A–3DGo) and BK-treatment (Figs. 3E–3HGo) conditions for 1 (BK-control, Figs. 3A–3DGo; BK treatment, Figs. 3E–3HGo), 3 (data not shown), and 10 (data not shown) min, double-labeling p-ERK1/2 with PGP 9.5, a marker for peripheral nerve fibers, was detected in nerve fibers and varicosities of the dentin-pulp complex.


Figure 3
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  		Figure 3. Immunohistochemical co-localization of PGP 9.5 and p-ERK1/2 in nerve fibers and nerve endings of the dentin-pulp complex. The nuclear DRAQ5 staining was detected in odontoblasts and pulp stroma cells after 1 min of BK-control (A) and BK-treatment (e) conditions. Immunohistochemical co-localization of green reaction product (DyLightTM 488) for PGP 9.5 (B, F; arrows) and red reaction product (DyLightTM 549) for p-ERK1/2 (C, G; arrows) in nerve fibers and varicosities of the dentin-pulp complex after 1 min BK-control (A-D) and BK-treatment (e-H) conditions. The double staining appears yellow for BK-control (D; double arrows) and after 1 min of BK stimulation (H; double arrows). Analyses of the double-labeled sections and images were acquired on an LSM510 laser-scanning confocal microscope. Bar: A-H = 10 µm.

 
Effects of BK on p-ERK1/2 in Nerve Terminals of the Dentin-Pulp Complex after 1, 3, and 10 min of BK Treatment
In control sections, nerve terminals revealed a decrease in staining intensities of p-ERK1/2 in nerve fibers of the dentin-pulp complex from 1 min (Fig. 4AGo), 3 min (Fig. 4CGo), to 10 min (Fig. 4EGo).


Figure 4
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  		Figure 4. The activation of p-ERK1/2 in nerve fibers and nerve terminals of the rat molar after 1, 3, and 10 min of BK treatment. In comparison with the controls for 1 min (A; G = 0.1217 ± 0.0265, asterisk) and 3 min (C; G = 0.0833 ± 0.0234, asterisk), the number of p-ERK1/2-stained nerve fibers (arrows) in the dentin-pulp complex was significantly increased at 1 (B; G = 0.2400 ± 0.0187, double asterisk) and especially at 3 (D; G = 0.2670 ± 0.0620, double asterisk) min after BK treatment. Double arrows reveal labeling for p-ERK1/2 only in a subpopulation of odontoblasts (C). Although nerve fibers revealed a time-dependent decrease in staining intensities of p-ERK1/2 from 1 (A) min to 10 (e) min in controls, the number of nerve fibers (arrows) was unchanged by comparison with nerve fiber number in the 10-minute control (E; G = 0.0545 ± 0.022) and 10-min BK-stimulation (F; G = 0.0543 ± 0.020) conditions. Bar: A-F = 120 µm; cp = coronal pulp, o = odontoblasts, pd = predentin, d = dentin. The histogram is defined as G.

 
In comparison with the control sections (Figs. 4A, 4GGo), the number of p-ERK1/2 immunoreactive nerve fibers of the dentin-pulp complex increased significantly at 1 min after BK treatment (Figs. 4B, 4GGo). In a subpopulation of odontoblasts, p-ERK1/2 was detected (Figs. 4C, 4GGo). Compared with the nerve fiber number of the control sections (Figs. 4C, 4GGo), the number of p-ERK1/2 immunoreactive nerve fibers and nerve terminals in the dentin-pulp complex was markedly increased at 3 min after BK treatment (Figs. 4D, 4GGo). In the dentin-pulp complex, no significant differences in the number of stained nerve fibers were detected at 10 min under control conditions (Figs. 4E, 4GGo), as well as after 10 min of BK treatment (Figs. 4F, 4GGo).

No stained cells or nerve fibers were detected in the immunohistochemical controls (Appendix 8).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The immunohistochemical detection of p-ERK1/2 in nerve fibers of the perfused sections provides information about only the steady-state or constitutive protein localization of p-ERK1/2, while organ bath experiments demonstrate the local protein activation of p-ERK1/2 in nerve fibers of the dentin-pulp complex after BK treatment. The local increase of p-ERK1/2 in nerve terminals of the dentin-pulp complex contributes to BK-induced non-transcriptional modulation of ERK1/2 in nerve endings, producing peripheral sensitization in the dentin-pulp complex.

The proteins which are needed in the axon and synaptic terminals are synthesized in the cell body and transported along the axon in membranous organelles or protein complexes (Hirokawa and Takemura, 2005). The cell bodies of the p-ERK1/2-positive axons of the dentin-pulp complex are located in the trigeminal ganglion and/or superior cervical, oticum, and pterygopalatinal ganglia. The cytoplasmic local levels of p-ERK1/2 in nerve fibers of the dentin-pulp complex were increased after BK treatment up to 3 min and decreased after 10 min. When gene transcription was increased in dorsal horn neurons, the expression of ERK1/2 occurred over a longer time-course (> 24 hrs) (Ji et al., 2002), while the phosphorylation-mediated activation of ERK1/2 in nerve fibers of the dental pulp occurred with a short latency (< 10 min). Therefore, we hypothesized that the activation of ERK1/2 in nerve fibers of the dentin-pulp complex must be mediated by local phosphorylation. The local activation of ERK in nerve terminals of the dentin-pulp complex may be explained by post-translational phosphorylation of ERK1/2 in cytoplasm via key receptors and channels in response to BK.

The decreased staining intensities of p-ERK1/2 in nerve fibers from 1 min to 10 min in controls may be dependent on the alteration of phosphorylation under the organ bath conditions. In comparison with the controls of one-, three-, and 10-minute conditions, the number of p-ERK1/2-stained nerve terminals increased after 1 and 3 min of BK treatment, while the number of nerve fibers was unchanged after 10 min of BK treatment. These results are compatible with a selective local effect of BK on the phosphorylation of ERK1/2 in nerve fibers of the dentin-pulp complex in a time-dependent manner. Intraplantar injection of capsaicin has been shown to increase p-ERK1/2-positive nerve terminals in the epidermis in 2 min (Dai et al., 2002). Inhibition of the ERK1/2 by MEK inhibitors attenuates heat hyperalgesia by capsaicin and mechanical hyperalgesia by epinephrine (Aley et al., 2001; Dai et al., 2002). Therefore, it could be presumed that BK leads to a time-dependent activation of the ERK1/2 in nerve terminals of the dentin-pulp complex, inducing an increase in the sensitization of nociceptors in the dentin-pulp complex. It is likely that the activation of ERK1/2 in nerve fibers of the dentin-pulp complex may be mediated by local phosphorylation through upstream kinases MEK1/2.

BK receptors have been localized in sensory neurons, and antagonists of BK have analgesic actions (Steranka et al., 1988). BK is a potent algogenic substance (Walker et al., 1995) and exerts its physiological effects via G-protein-linked B1 and B2 receptors (Regoli and Barabe, 1980). In the dental pulp, BK evokes neurotransmitter release via a B2-dependent mechanism (Goodis et al., 2000). The hypersensitivity of TRPV1 and TTXR following stimulation of BK underlies the induction of peripheral sensitization (Julius and Basbaum, 2001). It is possible to speculate that BK activates the B2 receptors on nociceptor terminals in the dentin-pulp complex, leading to the local protein activation of ERK1/2. It may be suggested that the activated ERK1/2 increases the sensitivity of TRPV1 and TTX-resistant sodium channels Nav1.8/1.9, inducing peripheral sensitization (Julius and Basbaum, 2001; Ji et al., 2002) in the dentin-pulp complex.

BK sensitizes nociceptor peripheral terminals, reducing the pain threshold (Wang et al., 2006). In addition to the decreases in the threshold of transduction ion channels to external stimuli (Sugiura et al., 2002; Wang et al., 2006), BK alters the threshold and kinetics of voltage-gated sodium channels (Amaya et al., 2006; Wang et al. 2006) and reduces activity in voltage-gated K+ channels (Oh and Weinreich, 2004; Wang et al., 2006). In a time-dependent manner, BK-induced local activation of ERK1/2 may modulate stimulus-transducing ion channels and voltage-gated ion channels to sensitize the transduction and enhance the excitability of the nociceptors in the dentin-pulp complex. BK-dependent phosphorylation of ERK1/2 in nerve terminals can reduce the action potential threshold in sensory neurotransmission in the dentin-pulp complex. This suggestion is supported by the fact that peripheral sensitization and inflammation reduce action potential duration in nociceptors (Bhave and Gereau, 2004).

It is concluded that BK sensitizes peripheral nociceptor terminals through the local phosphorylation of ERK1/2 in the dentin-pulp complex. In a time-dependent manner, BK activates ERK1/2 in a non-transcriptional manner to reduce the pain threshold of nociceptor terminals, producing peripheral sensitization in the dentin-pulp complex.


   ACKNOWLEDGMENTS
 
This study was supported by the Forschungskommission of the Heinrich-Heine-University of Düsseldorf. The technical assistance of Ch. Hoffmann, J. Kozlowski, and E. Janßen is gratefully appreciated.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/12/1149/DC1.

Received for publication November 5, 2007. Revision received August 9, 2008. Accepted for publication September 23, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 87, No. 12, 1149-1154 (2008)
DOI: 10.1177/154405910808701202
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