Free
Meeting Abstracts  |   October 1999
Local Anesthetics Inhibit Muscarinic Receptor-mediated Activation of Extracellular Signal-regulated Kinases in Rat Pheochromocytoma PC12 Cells 
Author Affiliations & Notes
  • Zhiming Tan, M.D.
    *
  • Shuji Dohi, M.D., Ph.D.
  • Kenji Ohguchi, Ph.D.
  • Shigeru Nakashima, M.D., Ph.D.
    §
  • Yoshinori Nozawa, M.D., Ph.D.
  • *Postgraduate Student, Department of Anesthesiology and Critical Care Medicine. †Professor and Chair, Department of Anesthesiology and Critical Care Medicine. ‡Postgraduate Student, Department of Biochemistry. §Associate Professor, Department of Biochemistry. ∥Professor and Chair, Department of Biochemistry.
Article Information
Meeting Abstracts   |   October 1999
Local Anesthetics Inhibit Muscarinic Receptor-mediated Activation of Extracellular Signal-regulated Kinases in Rat Pheochromocytoma PC12 Cells 
Anesthesiology 10 1999, Vol.91, 1014. doi:
Anesthesiology 10 1999, Vol.91, 1014. doi:
THE action of local anesthetics is thought to be principally through electrophysiologic currents that travel through ion channels that are important for the clinical efficacy of the drugs. 1 It is generally accepted that local anesthetics exert their anesthetic and toxic effects by inhibiting voltage-gated Na+channels, yet unknown mechanisms may be involved. Ion channels are regulated by protein phosphorylation, 2 which is an important mechanism in controlling cellular functions. 3 Recent reports indicate that local anesthetics could interact with membrane phospholipids and proteins and then affect various cellular activities. 1 Furthermore, local anesthetics are reported to interfere with or modulate some important biochemical signaling molecules, such as acetylcholine receptors (AChR), 4 guanosine 5′-triphosphate–binding proteins (G proteins), 5 protein kinase C (PKC), 6,7 and adenosine 3′,5′-cyclic monophosphate. 8 Despite such recent findings, the molecular mechanism underlying local anesthetic actions is not completely understood.
The mitogen-activated protein kinase (MAPK) cascades were recently identified in mammalian cells and play important roles in cellular signal transduction. 9 Exposure of cells to proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of MAPK. 9 In mammalian cells, p44MAPK and p42MAPK, now known as ERK1 and ERK2, respectively, are the typical and best studied members of the MAPK family and are activated by phosphorylation on serine and threonine residues by MAPK kinase. 10 The stimulation of ion channels or receptors on the surface of membranes initiates a sequence of activation of PKC, Ras, Raf, and MAPK kinase, which in turn activates ERK. 10 Recently, several studies were performed to determine whether ERK plays a role in anesthesia-related phenomena, such as the effects of opioids in human neuroblastoma cells 11 and alcohol action in PC12 cells. 12 Rat pheochromocytoma PC12 cells contain Na+, K+, and Ca2+channels and several membrane-bound receptors, including muscarinic and nicotinic ACh receptors, 13 and have been used widely to investigate signal transduction.
Inhibition of muscarinic signaling has been suggested to explain some states of general anesthesia. 14 We also know that muscarinic AChRs (mAChRs) are widely distributed in the peripheral and central neuronal systems and play a role in motor function and the processing of sensory information in the human spinal cord. 15 Although the mechanisms for local anesthetic blockade of nervous conduction have been established, the mechanism of epidural and spinal anesthesia is unknown and may be more complex than simply the inhibition of Na+channels. 1 To identify the molecular mechanisms responsible for the actions of local anesthetics, we evaluated the effects of local anesthetics on muscarinic receptor-induced protein tyrosine phosphorylation and ERK activation in PC12 cells. Because we found that local anesthetics inhibit carbamoylcholine chloride (carbachol)–induced ERK activation, but not 4β-phorbol 12-myristate 13-acetate (PMA)–induced activation, we also evaluated the effects of tetrodotoxin, an Na+channel blocker; veratridine, an activator of Na+channels; and ouabain, an Na+–K+pump inhibitor, on CCh-induced ERK activation.
Materials and Methods 
Materials 
Procaine hydrochloride, tetracaine hydrochloride, lidocaine hydrochloride, bupivacaine hydrochloride, atropine sulfate salt, tetradotoxin, veratridine, ouabain, calyculin A, carbachol, and PMA were purchased from Sigma Chemical Company (St. Louis, MO). Pirenzepine dihydrochloride and 4-diphenyacetooxy-1,1-dimethylpiperidinium, methiodide, and pertussis toxin were obtained from Research Biochemicals International (Natick, MA). Dulbecco modified Eagle medium and horse serum were from Life Technologies (Grand Island, NY). Fetal bovine serum was from Nippon Bio-supply Center (Tokyo, Japan). Anti-phosphotyrosine mouse monoclonal antibody was from Upstate Biotechnology Incorporated (Lake Placid, NY). Anti-ERK mouse monoclonal antibody was from Affiniti Research Products Limited (Nottingham, UK). The goat anti-mouse immunoglobulin G horse-radish peroxidase-coupled secondary antibody and the enhanced chemiluminescence system used for Western blot analysis were obtained from Amersham Life Science (Buckinghamshire, UK). Other reagents were of the highest quality available.
Cell Culture of PC12 Cells 
A PC12 cell line was supplied by Dr. Y. Sugimoto (Shirakawa Institute of Animal Genetics, Fukushima, Japan). Monolayer cultures of the cells were maintained in 100-mm-diameter tissue culture dishes in Dulbecco modified Eagle medium supplemented with 10%(vol/vol) fetal bovine serum and 5%(vol/vol) horse serum in a humidified atmosphere containing of 5% carbon dioxide at 37°C. Stock cultures were subcultured routinely at a cell density of 2–3 × 106/dish at least once a week, and culture media were renewed every 2 days.
Western Blot Analysis of Protein Tyrosine Phosphorylation and Extracellular Signal-regulated Kinase Activation in PC12 Cells 
PC12 cells were subcultured in 60-mm-diameter tissue culture dishes at 1 × 106cells/dish and grown for 4 days. The cells were washed twice with 2 ml buffer A (25 mM Hepes, p  H 7.4, 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, and 1 mg/ml bovine serum albumin) and preincubated in 3 ml buffer A with or without local anesthetics or Na+current–modifying reagents (tetrodotoxin, veratridine, and ouabain) at 37°C for 10 min. The cells were stimulated with 1 mM carbachol or 200 nM PMA at 37°C for durations indicated in each experiment. The reaction was terminated by aspiration of the reaction buffer and washing twice with 2 ml ice-cold phosphate-buffered saline (8% NaCl, 0.2% KCl, 2.88% Na2HPO4· 12H2O, 0.2% KH2PO4, p  H 7.4). The washed cells were scraped quickly into 120 μl RIPA buffer (10 mM Tris-HCl, p  H 7.4, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulfronyl fluoride, 10 μg/ml leupeptin, 1 mM Na3VO4, 10 mM NaF, and 0.1 mM Na2MoO4) and transferred to a microcentrifuge tube. After incubation on ice for 30 min, the suspension was centrifuged at 13,000g  for 20 min to obtain the cell extract. Seventy micrograms of protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) and transferred electrophoretically to a nitrocellulose membrane. After the membranes were blocked with TBS-T (10 mM Tris-HCl, p  H 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 2% bovine serum albumin, membranes were incubated with anti-phosphotyrosine antibody or anti-ERK antibody at room temperature for 90 min and then with the goat anti-mouse immunoglobulin G horseradish peroxidase–coupled secondary antibody at room temperature for 60 min. Detection was performed using an enhanced chemiluminescence system. The density of protein bands was analyzed using a densitometer (Densitograph; Atto Corporation, Tokyo, Japan).
Statistical Analyses 
Data are presented as the mean ± SD from three experiments. Differences between values were evaluated using analysis of variance; when P  < 0.05, differences were considered significant.
Results 
Tyrosine Phosphorylation of Cellular Proteins and Extracellular Signal-regulated Kinase Activation in Response to Carbachol in PC12 Cells 
Treatment of cells with 1 mM carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins with approximate molecular weights of 111, 91, 84, 74, 65–70, 44, and 42 kd. Among these, two phosphoproteins with molecular masses of 44 and 42 kd were most distinct, and phosphorylation of both proteins reached a peak 2 min after stimulation of carbachol (fig. 1A). These two proteins were identified as active forms of ERK1 and ERK2 by Western blot analysis using anti-ERK antibody. As shown in figure 1B, two bands of ERKs were presented as doublets 2–15 min after carbachol stimulation. Tyrosine-phosphorylated bands of 44- and 42-kd proteins were overlapped with upper bands of ERKs doublets, respectively (figs. 1A and 1B). As determined by densitometric analysis, active forms of ERKs reached the maximum (about 20 times more than nonstimulated levels for ERK2 and 10 times more than nonstimulated levels for ERK1) at 2 min and returned to the basal level 15 min after stimulation (fig. 1C).
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
×
Such carbachol-induced tyrosine phosphorylation of 44- and 42-kd proteins was blocked completely by preincubation with 50 μM atropine. This indicates that carbachol-induced activation of ERK1 and ERK2 was through the mAChRs in PC12 cells (figs. 2A and 2B). The mAChR-mediated ERK activation was insensitive to pertussis toxin (fig. 3A). Pretreatment of PC12 cells with pertussis toxin (10 ng/ml) for 21 h failed to suppress the carbachol-induced activation of ERK1 and ERK2. To determine which subtype of mAChRs was involved, the cells were pretreated with 10 μM pirenzepine, the M1receptor antagonist, and with 10 μM 4-diphenyacetooxy-1,1-dimethylpiperidinium, the M3receptor antagonist. The carbachol-induced activation of ERKs was blocked completely by 4-diphenyacetooxy-1,1-dimethylpiperidinium but not by pirenzepine (fig. 3B).
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
×
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
×
Effects of Local Anesthetics on Carbachol-induced Protein Tyrosine Phosphorylation in PC12 Cells 
In PC12 cells preincubated with 1–10 × 10−4M procaine for 10 min, carbachol-induced protein tyrosine phosphorylation decreased significantly. Procaine suppressed tyrosine phosphorylation of ERKs in a concentration-dependent manner and almost completely abolished tyrosine phosphorylation of these proteins at 5 × 10−4M (fig. 4). Pretreatment of PC12 cells with tetracaine, lidocaine, and bupivacaine (5 × 10−4M) also inhibited carbachol-induced tyrosine phosphorylation of ERKs (fig. 5).
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
×
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
×
Inhibitory Effects of Local Anesthetics on Carbachol-induced Extracellular Signal-regulated Kinase Activation 
Phosphorylation of ERK1 and ERK2 was prevented by procaine in a dose-dependent manner (fig. 6A). Some significant effect (P < 0.05) was apparent with 10−4M procaine. Activation of these ERKs was almost completely suppressed in the presence of 5 × 10−4M procaine in PC12 cells (figs. 6B and 6C). The presence of tetracaine, lidocaine, and bupivacaine at 5 × 10−4M similarly inhibited the activation of both ERKs (fig. 7). Although procaine and tetracaine appeared to be more potent than lidocaine and bupivacaine, the effects of each local anesthetic did not differ significantly (P  > 0.05) at 5 × 10−4M.
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
×
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
×
Effects of Procaine on 4β-Phorbol 12-Myristate 13-Acetate–induced Extracellular Signal-regulated Kinase Activation 
A commonly used activator of PKC as a receptor-bypass stimuli, PMA caused activation of ERK1 and ERK2 and peaked 15 min after stimulation in PC12 cells (data not shown). We then considered whether procaine exerted inhibitory effects on PMA-induced ERK activation. Although 5 × 10−4M procaine almost completely suppressed carbachol-induced ERK activation (fig. 8A), it did not have any effects on PMA-induced ERK activation (fig. 8B).
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
×
Effects of Na+Current–modifying Reagents on Carbachol-induced Extracellular Signal-regulated Kinase Activation 
Carbachol-induced ERK activation was not affected by preincubating PC12 cells with tetrodotoxin or veratridine at 1 × 10−6M (fig. 9A). In addition, at 5 ×10−3M ouabain also had no effects on the carbachol-induced activation of ERKs (fig. 9B).
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
×
The Effects of Procaine on the AlF4-induced Extracellular Signal-regulated Kinase Activation and the Effect of Protein Phosphatase on Procaine-inhibited, Carbachol-induced Extracellular Signal-regulated Kinase Activation 
Because we knew that the combination of NaF plus AlCl3(presumably acting as AlF4) can directly activate G proteins, 16 PC12 cells were exposed to NaF plus AlCl3to determine whether the ERK cascade downstream of the G proteins in PC12 cells is influenced by procaine. AlF4caused a time-dependent activation of ERK1 and ERK2 (data not shown). However, 5 × 10−4M procaine did not affect the AlF4-induced ERK activation (fig. 10A). In addition, pretreatment of PC12 cells with 10 nM calyculin A, a potent phosphatase inhibitor, did not modify the effects of procaine on carbachol-induced ERK activation (fig. 10B).
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4−-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
×
Discussion 
The molecular mechanisms underlying local anesthetic action for neuronal activities are not well understood. We have presented data showing that carbachol induces ERK1 and ERK2 activation that is mediated by the activation of M3mAChR in PC12 cells and that procaine inhibits carbachol-mediated tyrosine phosphorylation and ERK activation in a concentration-dependent manner. Although tetracaine, lidocaine, and bupivacaine similarly inhibited carbachol-induced tyrosine phosphorylation and ERK activation, neither Na+channel modifiers (such as tetrodotoxin, an Na+channel blocker, or veratridine, an Na+channel activator) nor ouabain, an Na+–K+pump inhibitor, affected ERK activation. The inhibitory effects of local anesthetic were not observed in both PMA-induced ERK activation and AlF4-induced ERK activation. In addition, a potent phosphatase inhibitor did not modify the effects of procaine on the carbachol-induced ERK activation. Therefore, the site(s) of inhibition of local anesthetics is probably located at mAChR or an interface between the receptors and G protein (fig. 11), rather than in the downstream of G protein. The ERK signaling cascade was recently shown to play an important role in the receptor-mediated signaling processes in neuronal cells. 10,11 The current results would provide an important clue for better understanding the molecular mechanism of the local action of an anesthetic.
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
×
The MAPKs are a family of serine–threonine protein kinases that are activated in response to various stimuli and play important roles in cellular signal transduction. 9 Among three major MAPK cascades, ERK, Jun N-terminal kinase/stress-activated protein kinase, and p38, which was identified recently, 10 the ERK studied in the current investigation has been shown to be involved in development, proliferation, plastic functions such as long-term potentiation, 17 and apoptosis. 18 PC12 cells are stimulated with carbachol via  heterotrimetric G protein–coupled mAChR, 19,20 followed by the direct binding of the adapter protein Grb2 to Sos. 10 Then the Grb2–Sos complex activates the nucleotide exchange activity on Ras, which leads to activation of the Raf–ERK pathway. 10 In addition to this pathway, the Ras-independent pathway may involve PKC. 10 
Phospholipase C activated by heterotrimeric G protein acts on phosphatidylinositol 4,5-bisphosphate to produce inositol(1,4,5)-triphosphate and diacylglycerol, both of which serve as second messengers to mobilize calcium and activate PKC, respectively. 21,22 Protein kinase C could be involved in the ERK activation by Raf phosphorylation on serine residues. 23 Our current findings that carbachol induced tyrosine phosphorylation of p42- and p44-kd proteins and ERK activation in PC12 cells correspond with results observed in oligodendrocytes, the myelin-producing cells of the central nervous system. 24 Atropine and 4-diphenyacetooxy-1,1-dimethylpiperidinium, an M3mAChR antagonist, blocked concentration-dependent MAPK activation induced by carbachol. Activation of ERKs is implicated in the transmission of the signal for mAChR. 24–26 Although it is not clear whether the inhibition of M3mAChR-mediated tyrosine phosphorylation and ERK activation with procaine, lidocaine, bupivacaine, and tetracaine at clinically relevant concentrations is related to their anesthetic action, to toxic side effects, or both, many pharmacologic properties of local anesthetics can be attributed to their actions on signal transduction molecules.
Local anesthetics are known to bind to mAChRs 27 and nicotinic AChRs. 1 With open channels, anesthetic binding to the AChR occurs immediately after agonist stimulation. 1,27 Cohen-Armon et al.  19,20 have found with membrane prepared from brain that the binding of agonists to mAChRs and to the Na+channels in the open state is a coupled event mediated by G proteins. When an agonist-like carbachol binds to its specific receptor, mAChR, the G protein mediates the activity of transmembrane channels through which ions flow, leading to changes in transmembrane voltage that could trigger signal transmission, and thus signal transduction molecules could be altered. Inhibitors such as procaine, as a cationic inhibitor of the AChR, can bind to the inhibitory site before the channel opens and regulates the ion permeability of the AChR. 1,27 Local anesthetics can allosterically interact with mAChR, 28,29 and it has been reported that lidocaine interacts with primary and allosteric recognition sites on mAChR. 29 After we plotted a curve fit for Hill equation, we found that the Hill coefficient for the procaine concentration–inhibition relation was 2.4 (fig. 6D), it can be assumed that the inhibition of local anesthetic on ERK activation involves multiple sites of action. The current observations may suggest that suppression of mAChRs would be involved, at least in part, in the local anesthetic inhibition of carbachol-induced ERK activation.
Modulation of Na+channels by PKC is likely to have important effects on signal transduction and synaptic transmission in the central nervous system. 2 The action of ion channels and Na+–K+pumps, which are closely associated with PKC activity, has a central role in the regulation of neuronal excitability, so PKC has been considered as a major site of signal transduction molecules for anesthetic action. 6,30 The increased activity of PKC modulates neuronal signal transduction by phosphorylation of several membrane proteins, including voltage-dependent Na+and other ion channels. 31 The phosphorylation of Na+channels (α- subunits) could occur by altering PKC activity. 32 Although PKC-activated Raf is thought to be Ras independent, stimulation of PKC in COS cells leads to Ras activation followed by formation of the Ras–Raf-1 complex, 33 suggesting that PKC could activate ERKs by a mechanism distinct from that initiated by mAChR stimulation by carbachol. Activation of ERKs via  the M3mAChR subtype, such as M1and M5, is independent of intracellular or extracellular Ca2+but depends in part on PKC. 26 
The effects of local anesthetics on PKC, however, are complicated. In a recent in vivo  study, Nivarthi et al.  6 found that the PKC activity of the spinal cord was increased with clinically relevant concentrations of intrathecal procaine and tetracaine. In in vitro  experiments, dibucaine and tetracaine competitively inhibit the binding of PMA to its receptor. 34 Unlike staurosporin, an inhibitor of PKC, neither 10−5to 10−3M ropivacaine, 10−3M bupivacaine, nor 1–3 × 10−3M lidocaine exerts any effects on the PMA-induced inhibition of phosphoinositide breakdown in human SK-N-MC neuroblastoma cells. 35 Because Na+channels in PC12 cells could be blocked completely by 5 × 10−4M procaine, 36 the fact that PMA-induced ERKs activation was not affected by procaine suggests that PMA-stimulated PKC activity remained intact in our experiments. Therefore, inactivation or activation of PKC, in the presence of local anesthetics, might not influence the effects on ERKs in any important way. Therefore, the effects of local anesthetics on carbachol-induced ERK activation is unlikely to be involved in the inhibition via  Na+channels or the PKC-dependent pathways at the concentration used in the current experiments.
We can only speculate why the Na+current-modifying reagents did not affect mAChR-mediated ERK activation. Several Na+transport pathways, such as the Na+channel and the Na+–K+pump, are implicated in changes in the intracellular Na+concentration. The membrane depolarization of the voltage-gated Na+channel affects G protein–coupled mAChR only if the voltage-gated Na+channels can be activated, 20 and the blockade of Na+channels by tetrodotoxin decreases muscarinic agonist binding to the receptors, whereas an Na+channel activator such as veratridine increases the binding. 19 
Furthermore, it has been reported that the tyrosine phosphorylation process seems to directly control the Na+–K+pump activity in the proximal convoluted tubule, 37 and veratridine stimulates phosphoinositide breakdown, which is inhibited by local anesthetic. 38 Therefore, we assumed that tetrodotoxin, veratridine, and ouabain could have some effect on muscarinic signal transduction of ERKs. However, we observed no effect in the presence of those Na+-modifying reagents. It is possible that such Na+-modifying reagents may not affect cellular events via  pertussis toxin–insensitive G proteins, such as carbachol-induced ERK activation. In human neutrophils, tetrodotoxin and veratridine have been reported not to affect PMA-induced O2generation, but eight local anesthetics, including procaine, suppress it in their concentration-dependent and lipid solubility–dependent manners. 39 
The application of carbachol reduces the peak Na+current without changing the voltage dependency of the channels, and the activation of mAChR strongly modulates Na+channel activity. 40 In addition, the channel-mediated Na+entry and Na+–K+pump activity are functionally interdependent, 41 and binding to mAChR is voltage dependent. 20 Therefore, it is conceivable that the stimulation by carbachol might have already altered the channel functions and Na+–K+-pump functions of PC12 cells, so neither tetrodotoxin, veratridine, nor ouabain may cause any change in mAChR-mediated events. Although we cannot completely exclude the possibility that Na+-modifying reagents could affect ERK activation, the inhibition of mAChR-induced ERK activation by local anesthetics seems likely to be independent of blockade of Na+currents and electrochemical gradients across the PC12 cell membrane.
It is unclear whether a potential interaction with M3mAChR plays any role in local anesthetic action or state. As shown in the current results and by findings of other investigations, M3mAChRs are coupled to a pertussis toxin–insensitive G protein and seem to regulate the activation of membrane-bound guanylyl cyclase. 14,42 The M3mAChRs have been reported to exist extensively in the spinal cord 43 and in brain regions, including the cerebral cortex, caudate nucleus, globus pallidus, the substantia nigra, and the hypothalamus, 44 and to play some role in neuronal cellular activities, such as N-methyl-D-aspartate receptor-mediated adenosine release in the cortex. 44 An activator of G protein, AlF4, caused time-dependent ERK1 and ERK2 activation, but 5 × 10−4M procaine did not affect the AlF4-induced activation (fig. 10A). Neither did a phosphatase inhibitor modify the effects of procaine on the carbachol-induced ERK activation. Therefore, these data may provide further evidence that the inhibition of muscarinic signaling by local anesthetics that we observed could exist in some specific sites of interaction with M3mAChR signaling molecules, probably at the mAChR, an interface between mAChR and G protein, or membrane lipid microdomains around the receptors and G-protein molecules, but this is unlikely because of the effect on G proteins per se  . The inhibition of M3mAChR-mediated ERK activation should be considered a convincing candidate for one target of local anesthetic action. Because little work has been done on the local anesthetic effects on ERK signaling, further studies are needed to determine the precise sites of local anesthetic action on the ERK signaling molecules.
In conclusion, the local anesthetics procaine, lidocaine, tetracaine, and bupivacaine, at their clinically relevant concentrations, inhibit carbachol-induced ERK activation, which is implicated in the transmission of signals via  M3mAChR-mediated cellular functions in PC12 cells. The target(s) that local anesthetics affect may be upstream of G proteins, including mAChR. Our findings may provide evidence that the inhibition of the ERK cascade is crucial for the action of local anesthetics. The lack of effect of Na+-current–modifying reagents on the muscarinic receptor-mediated ERK activation may suggest a great diversity of electrophysiologic and pharmacologic properties of local anesthetic action in signaling.
References 
References 
Butterworth JF, Stricharz GR: Molecular mechanisms of local anesthesia: A review. A NESTHESIOLOGY 1990; 72: 711–34
Numann R, Catterall WA, Scheuer T: Functional modulation of brain sodium channels by protein kinase C phosphorylation. Science 1991; 254: 115–8
Stryer L: Biochemistry, 4th ed. New York, WH Freeman, 1995, pp 244–7
Tigyi J, Tigyi G, Lilion K, Miledi R: Local anesthetics inhibit receptors coupled to phosphoinositide signaling in Xenopus oocytes. Eur J Physiol 1997; 433: 478–87
Hageluken A, Grunbaum L, Nurnberg B, Harhammer R, Schunack W, Seifert R: Lipophilic β-adrenoceptor antagonists and local anesthetics are effective direct activators of G-proteins. Biochem Pharmacol 1994; 47: 1789–95
Nivarthi RN, Grant GJ, Turndorf H, Bansinath MR: Spinal anesthesia by local anesthetics stimulates the enzyme protein kinase C and induces the expression of an immediate early oncogene, c-fos. Anesth Analg 1996; 83: 542–7
Trudell JR, Costa AK, Hubbell WL: Inhibition of protein kinase C by local anesthetics. Ann N Y Acad Sci 1990; 625: 743–6
Gordon LM, Dipple ID, Sauerheber RD, Esgate JA, Houslay MD: The selective effects of charged local anesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma. Neurotoxicology 1991; 12: 473–92
Malarkey K, Belham CM, Paul A, Graham A, Mclees A, Scott PH, Plevin R: The regulation of tyrosine kinase signaling pathways by growth factor and G-protein-coupled receptors. Biochem J 1995; 309: 361–75
Gutkind JS: The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1998; 273: 1839–42
Gutstein HB, Rubie EA, Mansour A, Akil H, Woodgett JR: Opioid effects on mitogen-activated protein kinase signaling cascades. A NESTHESIOLOGY 1997; 87: 1118–26
Roivainen R, Hundle B, Messing RO: Ethanol enhances growth factor activation of mitogen-activated protein kinases by a protein kinase C-dependent mechanism. Proc Natl Acad Sci U S A 1995; 92: 1891–5
Shafer TJ, Atchison WD: Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: A model for neurotoxicological studies. Neurotoxicology 1991; 12: 473–92
Durieux ME: Muscarinic signaling in the central nervous system. Recent developments and anesthetic implications (Review). A NESTHESIOLOGY 1996; 84: 173–89
Scatton B, Dubois A, Javoy-Agid F, Camus A: Autoradiographic localization of muscarinic cholinergic receptors at various segmental levels of the human spinal cord. Neurosci Lett 1984; 49: 239–45
Blackmore PF, Exton JH: Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon: Modulation by cAMP and phorbol myristate acetate. J Biol Chem 1986; 261: 11056–63
English JD, Sweatt JD: Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 1996; 271: 24329–32
Cano E, Mahadevan LC: Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 1995; 20: 117–22
Cohen-Armon M, Garty H, Sokolovsky M: G-protein mediates voltage regulation of agonist binding to muscarinic receptors: Effects on receptor-Na channel interaction. Biochemistry 1988; 27: 368–74
Cohen-Armon M, Sokolovsky M: Evidence for involvement of the voltage-dependent Na+channel gating in depolarization-induced activation of G-proteins. J Biol Chem 1993; 268: 9824–38
Takai Y, Kishimoto A, Kikawa U, Mori T, Nishizuka Y: Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophy Res Commun 1979; 91: 1218–24
Streb H, Irvine RF, Berridge MJ, Schulz I: Release of Ca2+from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 1983; 306: 67–8
Kolch W, Heidecker G, Kochs G, Hummel T, Vahidi H, Mischak H, Finkenzeller G, Marme D, Rapp UR: Protein kinase Cα activates RAF-1 by direct phosphorylation. Nature 1993; 364: 249–52
Larocca JN, Almazan G: Acetylcholine agonists stimulate mitogen-activated protein kinase in oligodendrocyte progenitors by muscarinic receptors. J Neurosci Res 1997; 50: 743–54
Lopez-Ilasaca M, Crespo P, Pekkici PG, Gutkind JS, Wetzker R: Linkage of G-coupled receptors to the MAPK signaling pathway through PI3-kinase γ. Science 1997; 275: 394–7
Wotta DR, Wattenberg EV, Langason RB, el-Fakahany EE: M1, M3and M5muscarinic receptors stimulate mitogen-activated protein kinase. Pharmacology 1998; 56: 175–86
Neher E, Steinbach JH: Local anesthetics transiently block currents through single acetylcholine receptor channels. J Physiol 1978; 277: 153–76
Lee NH, EL-Fakahany EE: Allosteric interactions at the m1, m2and m3muscarinic receptor subtypes. J Pharmacol Exp Ther 1991; 256: 468–79
Cohen-Armon M, Henis YI, Kloog Y, Sokolovsdy M: Interactions of quinidine and lidocaine with rat brain and heart muscarinic receptors. Biochem Biophys Res Commun 1985; 127: 326–32
Slater SJ, Kelly MB, Larkin JD, Ho C, Mazurek A, Taddeo FJ, Yeager MD, Stubbs CD: Interaction of alcohols and anesthetics with protein kinase Cα. J Biol Chem 1997; 272: 6167–73
Raymond LA, Blackstone CD, Huganir RL: Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity (Review). Trends Neurosci 1993; 16: 147–53
West JW, Numann R, Murphy BJ, Scheuer T, Catterall WA: A phosphorylation site in the Na+ channel required for modulation by protein kinase C. Science 1991; 254: 866–8
Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ: Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase. Science 1998; 280: 109–12
Uratsuji Y, Nakanishi H, Takeyama Y, Kishimoto A, Nishizuka Y: Activation of cellular protein kinase C and mide of inhibitory action of phospholipid-interacting compounds. Biochem Biophys Res Comm 1985; 130: 654–61
Martinsson T, Fowler CJ: Local anesthetics do not affect protein kinase C function in intact neuroblastoma cells. Life Sci 1993; 53: 1557–65
Olschewski A, Hempelmann G, Vogel W, Safronov BV: Blockade of Na+and K+currents by local anesthetics in the dorsal horn neurons of the spinal cord. A NESTHESIOLOGY 1998; 88: 172–9
Feraille E, Carranza ML, Rousselot M, Favre H: Modulation of Na+,K+-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule. J Physiol 1997; 498: 99–108
Tiger G, Fowler CJ: Further validation of a simple biochemical method for evaluation of local anesthetic effects at sodium channels. Methods Find Exp Clin Pharmacol 1996; 18: 431–5
Hattori M, Dohi S, Nozaki M, Niwa M, Shimonaka H: The inhibitory effects of local anesthetics on superoxide generation of neutrophils correlate with their partition coefficients. Anesth Analg 1997; 84: 405–12
Cantrell AR, Ma JY, Scheuer T, Catterall WA: Muscarinic modulation of sodium current by activation of protein kinase C in rat hippocampal neurons. Neuron 1996; 16: 1019–26
Sontheimer H, Fernandez-Marques E, Ullrich N, Pappas CA, Waxman SG: Astrocyte Na+channels are required for maintenance of Na+-K+-ATPase activity. J Neurosci 1994; 14: 2464–75
Flynn DD, Mash DC: Distinct kinetic binding properties of N-[3H]-methylscopolamine afford differential labeling and localization of M1, M2, and M3muscarinic receptor subtypes in primate brain. Synapse 1993; 14: 283–96
Hoglund AU, Baghdoyan HA: M2, M3and M4, but not M1, muscarinic receptor subtypes are present in rat spinal cord. J Pharmacol Exp Ther 1997; 281: 470–7
Semba K, White TD: M3muscarinic receptor-mediated enhancement of NMDA-evoked adenosine release in rat cortical slices in vitro. J Neurochem 1997; 69: 1066–72
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
Fig. 1. Carbachol-induced protein tyrosine phosphorylation and extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) The cells were stimulated with 1 mM carbachol for the indicated times. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. Carbachol induced rapid and transient increases in tyrosine phosphorylation of several proteins. The molecular mass markers, in kilodaltons, are indicated on the left. (  B  ) By using anti-ERK antibody, 44- and 42-kd phosphoproteins were identified as active forms of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  C  ) The density of bands of activated ERKs was measured by the densitometer (○ ERK1;• ERK2). Data represent the mean ±SD from three experiments. *  P  < 0.05  versus  0 min;**  P  < 0.0001  versus  0 min. 
×
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 2. Muscarinic receptor–mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERKs). PC12 cells were treated with the absence (−) or the presence (+) 50 μM atropine (Atro) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody or anti-ERK antibody were performed. (  A  ) The presence of atropine abolished tyrosine phosphorylation of 44- and 42-kd proteins. The molecular mass markers of p44 and p42 expressed in kilodaltons are indicated on the left. (  B  ) Atropine completely blocked the activation of ERK1 and ERK2. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
×
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
Fig. 3. The muscarinic acetylcholine receptor (mAChR) subtype involved in carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) Carbachol-induced ERK activation insensitive to pertussis toxin. The cells were treated with or without 10 ng/ml pertussis toxin (PTX) for 21 h and then stimulated with the presence (+) or the absence (−) of 1 mM carbachol for 2 min. (  B  ) The effects of mAChR antagonists on carbachol-induced ERK activation. The cells were stimulated with or without 1 mM carbachol in the presence (+) or the absence (−) of 10 μM pirenzepine (PZP) or 4-diphenyacetooxy-1,1-dimethylpiperidinium (4-DAMP). The antagonists were added 10 min before the addition of carbachol.  MATH-ERK1 and  MATH-ERK2 indicate activated ERK1 and ERK2. 
×
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
Fig. 4. The effects of procaine on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated with the indicated concentrations of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by procaine in a dose-dependent manner. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left. 
×
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
Fig. 5. The inhibitory effects of local anesthetics on carbachol-induced protein tyrosine phosphorylation in PC12 cells. The cells were treated in the presence (+) or the absence (−) of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-phosphotyrosine antibody were performed. The carbachol-induced protein tyrosine phosphorylation of 44- and 42-kd proteins was inhibited by the presence of each local anesthetic. The molecular mass markers of p44 and p42, expressed in kilodaltons, are indicated on the left with arrows. 
×
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
Fig. 6. The effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were preincubated with the indicated concentration of procaine (Pro) for 10 min and then stimulated in the presence (+) or the absence (−) of 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) Procaine dose dependently inhibited the activation of ERKs. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of (  B  ) ERK1 and (  C  ) ERK2 was measured by densitometric analysis and expressed as percentages of the result obtained in the absence of procaine. Data represent the mean ±SD from three different experiments. *  P  < 0.05  versus  0 × 10−4M;**  P  < 0.0001  versus  0 × 10−4M. (  D  ) The Hill plots of the procaine inhibition of the carbachol-induced ERK activation are shown. Y and 1−Y represent inhibited and noninhibited ERK activation, respectively. The Hill plots were made by computerized analysis of log [Y/1−Y]  versus  log procaine concentration, thereby yielding the Hill coefficients n = 2.4 for the effects of procaine on ERK1 and ERK2. 
×
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
Fig. 7. The inhibitory effects of local anesthetics on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro), tetracaine (Tetra), lidocaine (Lido), or bupivacaine (Bupi) for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. (  A  ) The activated forms of ERKs were prevented by the presence of each local anesthetic. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). (  B  ) The density of shifted bands (active forms) of ERK1 and ERK2 was measured using the densitometric analysis. Data represent the mean ± SD of three experiments. *  P  < 0.0001  versus  carbachol. 
×
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
Fig. 8. Comparison of the effects of procaine on carbachol- and 4β-phorbol 12-myristate 13-acetate (PMA)–induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. The cells were treated in the presence or the absence of 5 × 10−4M procaine (Pro) for 10 min and then stimulated with 1 mM carbachol for 2 min (  A  ) or 200 nM PMA for 15 min (  B  ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis with anti-ERK antibody were performed. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). The density of shifted bands (active forms) of ERKs by (  C  ) carbachol or (  D  ) PMA was analyzed by the densitometer, respectively. Data represent the mean ± SD of three experiments. *  P  < 0.05  versus  none;**  P  < 0.0001  versus  none;##  P  < 0.0001  versus  carbachol. 
×
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 9. The effects of (  A  ) tetrodotoxin (TTX), veratridine (VTD), and (  B  ) ouabain (Oua) on carbachol-induced activation of extracellular signal-regulated kinases (ERKs) in PC12 cells. The cells were treated in the presence or absence of 1 × 10−6M tetrodotoxin, 1 × 10−6M veratridine, or 5 ×10−3M ouabain for 10 min and then stimulated with or without 1 mM carbachol for 2 min. Extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two arrows indicate ERK2 (  MATH, active forms of ERK2). 
×
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4−-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
Fig. 10. The mechanisms of the effects of procaine on carbachol-induced extracellular signal-regulated kinase (ERK) activation in PC12 cells. (  A  ) There were no effects of procaine on the AlF4-induced ERK activation. The cells were treated with or without 5 × 10−4M procaine and then stimulated with 10 mM NaF plus 10 μM AlCl3for 30 min. (  B  ) There were no effects of calyculin A on the procaine inhibiting the carbachol-induced ERK activation. The cells were exposed to the presence (+) or the absence (−) of 10 nM calyculin A for 10 min and then 5 × 10−4M procaine was added. Ten minutes after procaine treatment, the cells were stimulated with 1 mM carbachol for 2 min. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-ERK antibody. The upper two arrows on the left indicate ERK1 (  MATH, active forms of ERK1) and the lower two indicate ERK2 (  MATH, active forms of ERK2). 
×
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
Fig. 11. A hypothetical scheme of the effects of local anesthetics on muscarinic receptor–mediated extracellular signal-regulated kinases (ERKs) in PC12 cells. The stimulation of muscarinic receptors in the cellular membranes initiates a sequence of activation of protein kinase C, Ras, Raf, and mitogen-activated protein kinase (MEK) that in turn activates ERKs. Local anesthetics perturb the cascade of the m3AChR-mediated ERK activation. In addition, the action of local anesthetics is independent of their effects on the voltage-dependent Na+channels. Inhibition of Na+–K+pumps does not affect the muscarinic acetylcholine receptor–mediated ERK activation. 
×