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Meeting Abstracts  |   December 1999
Dibucaine and Tetracaine Inhibit the Activation of Mitogen-activated Protein Kinase Mediated by L-type Calcium Channels in PC12 Cells 
Author Affiliations & Notes
  • Masumi Kansha, M.D.
    *
  • Taro Nagata, M.D.
    *
  • Kazuo Irita, M.D.
  • Shosuke Takahashi, M.D.
  • *Instructor. †Associate Professor. ‡Professor.
Article Information
Meeting Abstracts   |   December 1999
Dibucaine and Tetracaine Inhibit the Activation of Mitogen-activated Protein Kinase Mediated by L-type Calcium Channels in PC12 Cells 
Anesthesiology 12 1999, Vol.91, 1798. doi:
Anesthesiology 12 1999, Vol.91, 1798. doi:
SIGNALS initiated by neurotransmitter release are transferred to the nuclei of the postsynaptic cells to activate specific programs of gene expression, thus influencing synaptic functions. 1,2 
It has recently been reported that mitogen-activated protein (MAP) kinase (also known as extracellular signal reactive kinase [Erk]) might play an important role in linking membrane depolarization to gene expression in the postsynaptic neurons. 3,4 MAP kinase is highly expressed in the central nervous system, primarily in such postsynaptic structures as neuronal cell bodies and the bases of the proximal dendrites. 5 MAP kinase has been shown to be activated by calcium influx through both N  -methyl-D-aspartate (NMDA) receptors and L-type Ca2+channels. 6,7 Membrane depolarization and the subsequent Ca2+influx stimulate MAP kinase;4,5,8,9 The Ca2+influx stimulates Ras activation; Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf; MEK phosphorylates and activates MAP kinase. The activated MAP kinase is translocated to the nucleus and induces the expression of genes by phosphorylating and activating such transcriptional factors as c-Myc or Elk1 (fig. 1). 3,10,11 
Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
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The purpose of this study is to investigate whether local anesthetics affect the MAP kinase pathway activated by depolarization in neuronal cells. The rat pheochromocytoma cell line PC12 is considered to be a useful model for studying the mechanisms for a variety of calcium-dependent signaling events in the nervous system. 8,12–15 Here, we show the effects of dibucaine and tetracaine on the activation of MAP kinase in PC12 cells.
Materials and Methods 
The MEK inhibitor (PD 98059), phosphospecific MAP kinase antibody, phospho-p44/p42 MAP kinase monoclonal antibody, phosphospecific Elk1 antibody, anti-Elk1 antibody, active Erk2, and a p44/p42 MAP kinase assay kit were purchased from New England BioLabs (Beverly, MA). Protein A-agarose and anti-Erk1, anti-Erk2, and anti–-c-Fos antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). w-Conotoxin GIVA was from the Peptide Institute (Osaka, Japan). Dibucaine, tetracaine, nifedipine, ionomycin, and adenosine triphosphate were purchased from Sigma (St. Louis, MO).
Cell Culture 
PC12 cells were maintained in RPMI 1640 medium (Roswell Park Memorial Institute, developed by Dr. G. Moore) supplemented with 10% horse serum, 5% fetal bovine serum, 40 U/ml penicillin G, and 100 μg/ml streptomycin.
Detection of MAP Kinase Activity 
PC12 cells were plated on 60-mm collagen-coated dishes (6 × 106cells/dish) and incubated for 6–12 h in a low-serum medium (0.25% fetal bovine serum and 0.5% horse serum). After pretreatment with dibucaine or tetracaine for 10 min, the cells were stimulated with either 50 mM KCl or 1 μM ionomycin for 2 min, washed once with cold phosphate-buffered saline and lysed in 200 μl cell lysis buffer containing 20 mM tris(hydroxymethyl)-aminomethane (TRIS; pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM (ethylenedioxy)diethyl-enedinitrilotetraacetic acid (EGTA), 1% Triton X-100 (Sigma), 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 50 μM 4-amidinophenyl-methanesulphenyl fluoride (APMSF), and 1 μg/ml leupeptin. The cells were then scraped off and transferred to microcentrifuge tubes and sonicated with 3-× 5-s bursts in a Branson sonifier at low power in an ice bath. After centrifugation at 6,000 g  for 15 min at 4°C, the supernatant was transferred to a new tube and incubated with phospho-p44/p42 MAP kinase monoclonal antibody (1:200 dilution), which only detects MAP kinase phosphorylated at both threonine 202 and tyrosine 204, overnight at 4°C with gentle rocking. These phosphorylation sites are critical for the MAP kinase activity. Protein A-agarose was added for an additional 3 h. The immunoprecipitate was obtained by centrifugation at 6,000 g  for 1 min at 4°C and was washed twice with 0.5 ml cell lysis buffer and twice with 0.5 ml kinase buffer containing 25 mM TRIS (pH 7.5), 5 mM β-glycerophosphate, 2 mM 1,4-dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2. The immunocomplexes were incubated with 1.2 μg Elk1 fusion protein, which is glutathione S-transferase fused to Elk1 codons 307–428, and 200 μM adenosine triphosphate in kinase buffer for 30 min at 30°C. The reaction was terminated by adding Laemmli buffer (3 × concentration). After boiling for 5 min, the mixture was centrifuged at 6,000 g  for 2 min at 4°C. The supernatant was then subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted with phosphospecific Elk1 antibody, which detects only Elk1 phosphorylated at serine 383.
Detection of MAP Kinase Pphosphorylation 
After stimulation with 50 mM KCl for 2 min, the cells were washed once with cold phosphate-buffered saline and lysed in radioimmunoprecipitation buffer containing 50 mM TRIS (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (Sigma), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 2 mM sodium vanadate, 50 mM NaF, 30 mM p-nitrophenyl phosphate, 40 μM 4-APMSF, 40 μM leupeptin, 1 μM pepstatin, and 30 μg/ml aprotinin. The cells then were incubated on ice for 20 min. The cell lysates were centrifuged at 6,000 g  for 20 min at 4°C and the supernatant was resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted with phosphospecific MAP kinase polyclonal antibody that detects MAP kinase phosphorylated at tyrosine 204.
Detection of cFos 
For c-Fos detection, PC12 cells (1 × 107cells/dish) were plated on collagen-coated dishes, stimulated with 50 mM KCl at 37°C for 60 min, washed with cold phosphate-buffered saline three times, and lysed in lysis buffer containing 10 mM TRIS (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 20 μM 4-amidinophenyl-methanesulphenyl fluoride, 30 μM leupeptin, and 30 μg/ml aprotinin. After centrifugation at 6,000 g  for 5 min at 4°C, the pellet was resuspended in 2 × Laemmli buffer, boiled for 5 min, and sonicated briefly. The presence of c-Fos was determined by Western blot analysis.
Western Blot Analysis 
The samples were subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and were electrotransferred to the nitrocellulose membrane. The membranes were incubated with primary antibodies either for 45 min at room temperature or overnight at 4°C. The blots were probed with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (IgG) antibody for 1 h. Antibody binding was detected using the enhanced chemiluminescence system (Amersham Pharmacia Biotechnology, Uppsala, Sweden).
Statistical Analysis 
Western blots were scanned into the program Adobe Photoshop 4.0 (San Jose, CA) using an OPAL Ultra scanner (Heidelberg Jpapan, Tokyo, Japan) and then analyzed with NIH Image version 1.6 software (National Institutes of Health, Bethesda, MD). The results are expressed as a percentage of the density of the band obtained after stimulation with potassium chloride (KCl) or ionomycin without pretreatment (positive control) and presented as the mean ± SEM. Differences were analyzed using an unpaired t  test. P  < 0.05 was considered to be significant. The data points in concentration-dependent inhibition of dibucaine and tetracaine on the MAP kinase activity were fitted according to a four-paramater logistic model described by De Lean et al.,  16 and the concentrations that inhibit 50% of the activity of positive control (IC50s) were derived from these fits.
Results 
MAP Kinase Activity Stimulated with Potassium Chloride Depolarization 
When the PC12 cells were depolarized by KCl, MAP kinase was activated transiently as shown in figure 2A. The activity reached a maximum at 2 min and then decreased within 5 min. The MAP kinase activation depended on the Ca2+influx through voltage-sensitive calcium channels. The pretreatment of the cells with either 1 mM EGTA or 5 μM nifedipine blocked the MAP kinase activation, whereas the pretreatment of the cells with 2 μM ω-conotoxin GIVA did not (figs. 2B and C). These findings, therefore, indicate that the activation of MAP kinase is mediated by L-type Ca2+channels but not by N-type Ca2+channels in PC12 cells.
Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
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Effects of Dibucaine and Tetracaine on MAP Kinase Activation Induced by Potassium Chloride 
Dibucaine and tetracaine suppressed the activation of MAP kinase stimulated with KCl in a dose-dependent manner (figs. 3A and B). The pretreatment of cells with dibucaine or tetracaine inhibited the activation of the MAP kinase. The IC50values for dibucaine and tetracaine were 17.7 ± 1.0 μM and 70.2 ± 1.2 μM, respectively (fig. 3C). The local anesthetics had no effect on the time dependence of MAP kinase activation (data not shown). The changes in the pH of the medium caused by adding dibucaine or tetracaine were not significant (approximately 0.05).
Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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Effects of Dibucaine and Tetracaine on MAP Kinase Phosphorylation Induced by Potassium Chloride 
To confirm that dibucaine and tetracaine affect the MAP kinase signaling pathway, we evaluated the effects of local anesthetics on MAP kinase phosphorylation induced by KCl. As shown in figure 4, pretreatment with dibucaine or tetracaine inhibited the phosphorylation of both Erk1 (44 kd) and Erk2 (42 kd). The IC50values for dibucaine to inhibit the phosphorylation of Erk1 and Erk2 were 7.6 ± 0.8 μM and 13.1 ± 2.1 μM, respectively, and the IC50values for tetracaine to inhibit the phosphorylation of Erk1 and Erk2 were 82.6 ± 13.1 μM and 101.3 ± 3.2 μM, respectively (fig. 4C). These values were comparable to those obtained from the experiment concerning MAP kinase activation.
Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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Effects of Dibucaine and Tetracaine on MAP Kinase Activation Induced by Ionomycin 
To evaluate the probable sites of action of dibucaine and tetracaine on MAP kinase pathway, we evaluated the effects of these local anesthetics on the activation of MAP kinase after the elevation of intracellular Ca2+using a Ca2+ionophore. If intracellular Ca2+was increased with 1 μM ionomycin, a transient activation of MAP kinase was also observed (fig. 5A). Pretreatment of cells with dibucaine or tetracaine suppressed the activation of MAP kinase induced by ionomycin (figs. 5B and C). However, the IC50values for dibucaine and tetracaine (62.5 ± 2.2 m and 330.5 ± 32.8 μM, respectively) were approximately four times higher than those obtained in inhibition of KCl-induced MAP kinase activation (fig. 3C)
Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
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Effects of Dibucaine and Tetracaine on c-Fos Induction 
KCl-induced c-Fos expression was inhibited by 1 mM EGTA or 5 mM nifedipine (fig. 6). Pretreatment of the cells with 50 mM MEK inhibitor (PD 98059) also suppressed the c-Fos induction, suggesting that MAP kinase may be involved in the signaling pathway that links Ca2+influx to gene expression. As shown in figure 7, dibucaine and tetracaine also inhibited the depolarization-induced c-Fos expression. The IC50values for dibucaine and tetracaine (concentrations that inhibit 50% of the c-Fos expression induced by KCl without pretreatment) were 16.2 ± 0.2 mM and 73.2 ± 0.7 mM, respectively (fig. 7C). According to the trypan blue exclusion test, more than 95% of the cells were viable after incubation with 20 mM dibucaine, 200 mM tetracaine, or 50 mM MEK inhibitor for 60 min.
Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
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Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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Discussion 
In this study, we showed that dibucaine and tetracaine inhibited the activation of MAP kinase and the expression of c-Fos induced by KCl in PC12 cells. Both the activation of MAP kinase and the expression of c-Fos induced by KCl depended on L-type Ca2+channels. Because MEK inhibitor blocked KCl-induced c-Fos expression, it appears that the calcium signal mediated by L-type Ca2+channels is transmitted, at least in part, to the nucleus through the MAP kinase pathway.
Converse et al.  17 reported that during spinal anesthesia, the mean concentration of tetracaine in human cerebrospinal fluid is 0.8–12 mg/dl; namely, 26.7–400 μM. 17 Therefore, the concentrations of the local anesthetics that inhibited MAP kinase activation induced by KCl seemed to be within the normal clinical ranges.
Although dibucaine and tetracaine inhibited the activation of MAP kinase induced by KCl and ionomycin, IC50values for dibucaine and tetracaine obtained from the experiment using ionomycin were higher than those obtained from the experiment using KCl. Because it has been reported that Ras is necessary to induce the activation of MAP kinase by ionomycin, 9 there is a possibility that dibucaine and tetracaine at lower concentrations primarily affect upstream of Ras in MAP kinase pathway: L-type Ca2+channels or yet unknown mechanisms between Ca2+and Ras.
Sugiyama and Muteki 18 reported that local anesthetics inhibit the L-type Ca2+channels in rat dorsal root ganglion cells, although the physiologic implications of this remain to be elucidated. In their report, the IC50values for dibucaine and tetracaine were 34 and 79 μM, respectively, which are similar to the IC50values for dibucaine and tetracaine in our study (17.7 and 70.2 μM, respectively).
In animal studies, a blockade of the L-type Ca2+channels was reported to induce antinociceptive effects. 19–24 However, the mechanisms causing these effects have yet to be clarified. Although L-type Ca2+channels are expressed in neurons, they do not seem to play a key role in neurotransmitter release. 25–27 L-type Ca2+channels are localized to the base of the dendrites and neuronal cell bodies but not to the synaptic terminals, 28,29 thus suggesting that they mediate calcium-dependent signaling events in the postsynaptic neurons. In studies using cultured cortical neurons, the basal expression of specific immediate early genes were blocked by L-type Ca2+channel antagonists and increased by L-type Ca2+channel agonists. 30,31 In cultured hippocampal neurons, cultured spinal-cord neurons, and adrenal cromaffin cells, L-type Ca2+channels activated by KCl induced the expression of brain-derived neurotrophic factor and proenkephalin genes. 32–34 These observations suggest that L-type Ca2+channels appear to play a critical role in the coupling of synaptic excitation and gene expression. Although a relatively minor component of the calcium current evoked by synaptic activation is attributed to the L-type Ca2+channels, 19 the calcium influxes caused by synaptic excitation may be highly localized and activate the specific calcium-dependent signaling pathways.
Recently, not only the elevation of intracellular calcium, but also the route of calcium entry has been reported to influence the expression of genes by activating the distinct regulatory elements in the promoter region. 35–37 For example, in cultured cortical neurons, the activation of L-type Ca2+channels, but not NMDA receptors, increases cell survival by the enhanced expression of brain-derived neurotrophic factor, 38 thus suggesting the importance of L-type Ca2+channels in the nervous system. It has been reported that the calcium influx via  receptors activates gene transcription mediated by serum response element (SRE), and calcium entry via  L-type Ca2+channels activates gene transcription mediated by both cyclic adenosine monophosphate response element and SRE. 34–36 The signaling pathway that leads to SRE-dependent gene transcription is considered to include the activation of MAP kinase. 35–37,39 
Another possibility to explain the difference in the concentrations of local anesthetics between KCl and ionomycin to inhibit the activation of MAP kinase is that the level of intracellular Ca2+elevation might be higher on stimulation with 1 mM ionomycin than with 50 mM KCl. Higher concentrations of Ca2+may induce a strong activation of MAP kinase: This strong activation might necessitate higher concentrations of local anesthetics for inhibition. But this seems unlikely. According to Delorme et al.  , 40 the peak intracellular Ca2+concentrations measured using quin 2 after the addition of 50 mM KCl, 10 μM ionomycin (in the presence of 5% fetal bovine serum and 10% horse serum), and 50 nM ionomycin (in the absence of serum) are similar, probably because of protein binding of ionomycin. In our experiments, the cells were stimulated in the medium containing 0.25% fetal bovine serum and 0.5% horse serum. So, the peak concentration of intracellular Ca2+after the addition of 1 μM ionomycin is not supposed to be much higher than that after the addition of 50 mM KCl. However, there might be differences in the distribution of Ca2+within the cells between the two stimuli. The upstream pathways to the MAP kinase are supposed to be multiple, 4 and the difference in the distribution of Ca2+might activate these different pathways, resulting in the difference in IC50values for local anesthetics on stimulation with KCl from ionomycin. Therefore, although dibucaine and tetracaine at clinical concentrations possibly affect L-type Ca2+channels and components between calcium and Ras in MAP kinase pathway, we cannot rule out the possibility that these local anesthetics might inhibit the MAP kinase directly. It has been reported that two homologous proteins of MAP kinase family, Erk1 and Erk2, with molecular masses of 44 and 42 kd, are expressed in the central nervous system. 41 Whereas Erk2 is widely expressed, Erk1 is observed in restricted regions. 42 Both of these MAP kinases are activated by phosphorylation by MEK, although the difference in the role of these MAP kinases is not well-known. In our findings, dibucaine and tetracaine inhibited the phosphorylation of both Erk1 and Erk2 during stimulation with KCl.
In conclusion, we showed that dibucaine and tetracaine at clinical concentrations inhibited the depolarization-stimulated MAP kinase activation and c-Fos induction mediated by L-type Ca2+channels. MAP kinase may play an important role in gene expression and in control of synaptic functions. Our findings suggest that the inhibition of MAP kinase pathway might be one of the potential sites for the actions of local anesthetics in the nervous system.
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Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
Fig. 1. The Ca2+influx through L-type voltage sensitive Ca2+channels activates the mitogen-activated protein (MAP) kinase signaling pathway. Synaptic transmission induces depolarization in the postsynaptic membrane and activates the L-type voltage sensitive Ca2+channels (L-VSCC). The elevation of intracellular Ca2+activates Ras by some as yet unknown mechanism. Ras transmits its signal to mitogen-activated/extracellular receptor–regulated kinase (MEK) through Raf. MEK phosphorylates and activates p44/p42 MAP kinase. The activated MAP kinase phosphorylates transcriptional factors, such as Elk1. The complex of phosphorylated Elk1 and the homodimer of serum response element (SRE) serum response factors binds to SRE and induces the transcription of c-Fos. Ionomycin elevates intracellular Ca2+independent of L-type voltage sensitive Ca2+channels and also activates MAP kinase pathway dependent on Ras activation. 
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Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
Fig. 2. (  A  ) The time course of the KCl-induced activation of MAP kinase. (  B  ) The effects of EGTA and calcium channel blockers on the KCl-induced activation of mitogen-activated protein (MAP) kinase. PC12 cells were stimulated with 50 μM KCl for the indicated periods of time or with KCl for 2 min at 37°C after pretreatment with 1 mM EGTA, 2 mM ω-conotoxin GIVA, or 5 mM nifedipine for 5 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different form positive control at  P  < 0.05. 
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Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 3. Inhibition of KCl-induced mitogen-activated protein (MAP) kinase activation by dibucaine and tetracaine. The cells were pretreated with dibucaine or tetracaine for 10 min and then stimulated with 50 mM KCl for 2 min at 37°C. The MAP kinase activity was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the MAP kinase activity. Activation is expressed as a percentage of the activation caused by 50 mM KCl or 1 μM ionomycin without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 4. Dibucaine and tetracaine inhibited the phosphorylation of mitogen-activated protein (MAP) kinase induced by KCl. The cells were stimulated with 50 mM KCl for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The phosphorylation of MAP kinase was detected as described in Materials and Methods. (  A, B  ) The blots were probed with phosphospecific MAP kinase antibody (  upper  ) and a mixture of anti-Erk1 and anti-Erk2 antibodies (  lower  ). Typical results of six independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of MAP kinase. Phosphorylation of Erk1 and Erk2 is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of six independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
Fig. 5. Time course of the activation of mitogen-activated protein (MAP) kinase induced by ionomycin and inhibition of ionomycin-induced MAP kinase activation by dibucaine or tetracaine. (  A  ) The cells were stimulated with 1 μM ionomycin for indicated periods of time at 37°C. The MAP kinase activity was detected as described in Materials and Methods. Typical results of three independent experiments are shown. (  B, C  ) The cells were stimulated with 1 μM ionomycin for 2 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. The MAP kinase activity was detected as described in Materials and Methods. Protein blots were probed with phosphospecific Elk1 antibody (  upper  ) and anti-Elk1 antibody (  lower  ). Typical results of six independent experiments are shown. 
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Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
Fig. 6. The effects of dibucaine, tetracaine, EGTA, nifedipine, and mitogen-activated/extracellular receptor–regulated kinase (MEK) inhibitor on the KCl-induced expression of c-Fos. The cells were incubated with 50 mM KCl for 60 min at 37°C after pretreatment with 20 μM dibucaine, 200 μM tetracaine, 1 mM EGTA, or 5 μM nifedipine for 10 min, and with 50 μM MEK inhibitor (PD 98059) for 30 min. The expression of c-Fos was detected as described in Materials and Methods. (  A  ) Protein blots were probed with anti-c-Fos antibody. The typical results of three independent experiments are shown. (  B  ) A densitmetric analysis of the expression of c-Fos. c-Fos expression is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. *Different from positive control at  P  < 0.05. 
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Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
Fig. 7. Dose-dependent effects of dibucaine of tetracaine on the expression of c-Fos induced with KCl. The cells were stimulated with 50 mM KCl for 60 min at 37°C after pretreatment with dibucaine or tetracaine for 10 min. c-Fos was detected as described in Materials and Methods. (  A, B  ) Protein blots were probed with anti–c-Fos antibody. Typical results of three independent experiments are shown. (  C  ) A densitmetric analysis of the phosphorylation of mitogen-activated protein kinase. Phosphorylation is expressed as a percentage of that caused by 50 mM KCl without pretreatment (positive control). Values are mean ± SEM of three independent experiments. The lower values at negative control (nc) represent the percentage of activity of nonstimulated cells. 
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