Involvement of the Mitogen-activated Protein Kinase Family in Tetracaine-induced PC12 Cell Death

A PC12 cell line was donated by Dr. Yoshinori Sugimoto (Shirakawa Institute of Animal Genetics, Fukushima, Japan). Tetracaine hydrochloride, procaine hydrochloride, lidocaine hydrochloride, bupivacaine hydrochloride, EGTA, and BAPTA-AM were purchased from Sigma Chemical Company (St. Louis, MO). BAPTA-AM was first diluted in dimethyl sulfoxide as a stock solution (10 mm) and added to culture media for 60 min before tetracaine treatment. Dulbecco modified Eagle medium and horse serum were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum was obtained from Nippon Bio-supply Center (Tokyo, Japan). PD98059 were obtained from Calbiochem (San Diego, CA). Phospho-ERK (Thr202–Tyr204) antibody, phospho–stress-activated protein kinases–JNK (Thr183–Tyr185) antibody, and phospho-p38 (Thr180–Tyr182) antibody were obtained from New England Biolabs (Beverly, MA). Caspase-3 antibody and poly(adenosine diphosphate–ribose) polymerase (PARP) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat antirabbit immunoglobulin, horseradish peroxidase-linked whole antibody, and enhanced chemiluminescence system were obtained from Amersham Life Science (Buckinghamshire, United Kingdom). Hoechst 33258 (bisbenzimide) staining dye was obtained from Wako, Inc. (Osaka, Japan). Other reagents were of the highest quality available.

Monolayer cultures of PC12 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 humidified atmosphere containing of 5% CO², at 37°C. Stock cultures were subcultured routinely at a cell density 2–3 × 10⁶per dish at least once a week, and culture media were renewed every 2 days.

PC12 cells were plated in 35-mm-diameter tissue culture dishes at 2 × 10⁵cells per dish and grown for 4 days and then exposed to the culture media containing indicated agents in humidified atmosphere containing of 5% CO², at 37°C for indicated times. After treatment, the cells were collected, washed twice with cold phosphate-buffered saline (PBS), and then suspended with 500 μl PBS. Cell death of PC12 cells was assessed by bright-field microscopy using a 0.5% trypan blue dye exclusion method. The death was determined by counting three randomly selected fields per sample. Three independent experiments were performed in triplicate on separate occasions with different cultures.

Lactate dehydrogenase (LDH) activity released from injured cells was assessed using a commercially available kit (Kyokuto, Tokyo, Japan) as described by the manufacturer. PC12 cells were plated in 35-mm-diameter tissue culture dishes at 2 × 10⁵cells per dish and grown for 4 days and then exposed to the culture media containing indicated agents in humidified atmosphere containing of 5% CO², at 37°C for indicated times. The cells in media were removed by centrifugation (900 g , 5 min), and LDH activity in the media was determined.

PC12 cells were washed twice with PBS at 37°C and fixed with 1% glutaraldehyde (in PBS) for 30 min at 4°C. After the cell suspension had been centrifuged, the pellet was postfixed in a solution of 2% OsO⁴in PBS buffer for 60 min at 4°C, then dehydrated through graded concentrations of acetone and critical-point dried from liquid carbon dioxide. The material was mounted, coated with gold, and viewed in a JSM-U3 scanning electron microscope (JEOL Ltd., Tokyo, Japan).

After treatment described above, the cells were collected, fixed with 1% glutaraldehyde for 30 min at 4°C, washed with PBS, and stained with 1 μm Hoechst 33258 for 5 min. The nuclei were analyzed under a nonconfocal fluorescence microscope (Olympus BX60; Olympus Ltd., Tokyo, Japan), with excitation at 360-nm ultraviolet illumination.

Cells were cultured on 100-mm-diameter dishes, and treated cells were collected in cold PBS. After centrifugation (1,500 g , 5 min), the pellet was resuspended in lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 1% Triton X-100) for 10 min at 4°C. The supernatant was saved after centrifugation (16,000 g , 20 min) and treated with DNase-free RNase (10 μg/ml) and proteinase K (0.1 mg/ml) for 2 h at 37°C. Genomic DNA was precipitated by the addition of 1:6 (vol/vol) of 5 m NaCl and isopropyl alcohol overnight at −20°C, pelleted, and redissolved in loading buffer. DNA samples were then size-fractionated on a 2% agarose gel.

PC12 cells were fixed with 1% glutaraldehyde in PBS buffer at 37°C for 30 min, washed twice with PBS, and then resuspended with 30% fresh glycerol in PBS overnight. Cells were frozen in Freon-12 and transferred to liquid nitrogen (−195°C). Frozen samples were fractured and shadowed with platinum-carbon in a freeze-fracture apparatus (Hitachi HF-1; Hitachi Ltd., Tokyo, Japan). Replicas were cleaned with hypochlorite solution and then examined with a JEM 1010 electron microscope (JEOL Ltd.).

PC12 cells were plated in 35-mm-diameter tissue culture dishes at 2 × 10⁵cells per dish and grown for 4 days. The cells were incubated with the culture media containing the cytoplasmic calcium indicator, Fluo-3 AM (3 μm; Nacalai Tesque, Inc., Kyoto, Japan) for 120 min. Treated cells were collected by centrifugation (900 g , 5 min) and suspended in 2 ml PBS. The fluorescence was measured using Versa Fluorometer (Bio-Rad, Tokyo, Japan) with excitation at 490 nm and emission at 510 nm.

PC12 cells were subcultured in 60-mm-diameter tissue culture dishes at 10⁶cells per dish and grown for 4 days. After treatment, the cells were washed twice in cold PBS and lysed with buffer A (10 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecylsufate [SDS], 150 mm NaCl, 1 mm EDTA, 0.5 mm phenylmethylsulgronyl fluoride, 10 μg/ml leupeptin, 1 mm Na³VO⁴, 10 mm NaF, and 1 mm Na²MoO⁴). After incubation for 30 min at 4°C, the suspension was centrifuged at 13,000 g  for 20 min to obtain the cell extract. Proteins were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to nitrocellulose membrane. After blocking of the membranes with TBS-T (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Tween 20) containing 2% bovine serum albumin, membranes were incubated with the indicated antibodies at room temperature for 90 min and then with the goat antirabbit immunoglobulin horseradish peroxidase-linked whole antibody at room temperature for 60 min. Detection was performed with enhanced chemiluminescence system.

The data are expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance, followed by the Fisher least significant difference test. Differences were considered to be statistically significant at P < 0.05.

PC12 cells were exposed to tetracaine at concentrations from 0.2 to 1 mm. Tetracaine induced PC12 cell death in a time- and concentration-dependent fashion (fig. 1A). More than 50% of cells were dead after treatment with 1 mm tetracaine for 4 h; the cell death was reached at 90% after 10 h of treatment. Similar to cell death, LDH released from cells treated with tetracaine for 10 h was also in concentration-dependent manner (figs. 1A and B). Using a scanning microscope, cellular morphology changes were observed in response to tetracaine (fig. 2). After treatment of 1 mm tetracaine for 4 h, some blebs arised from cell surfaces and microvilli vanished, and after 8 h, pseudopodia (budding) emitted off cells. Nuclei were examined by staining with Hoechest 33258 (fig. 3). In the presence of 1 mm tetracaine, the nuclei of dead cells showed condensation, and some appeared fragmented. DNA fragmentation was apparent by gel electrophoresis by 2 h after treatment with 1 mm tetracaine (fig. 4). Freeze-fracture electron micrographs showed that intramembranous particles, which are considered to represent integral membrane proteins, 29are markedly aggregated, and smooth intramembranous particle–free areas appeared on the protoplasmic fracture face in tetracaine-treated cell membrane, in contrast to untreated cell membrane, in which numerous intramembranous particles were randomly distributed (figs. 5A and B).

Caspase-3 has been identified as a key protease activated by cleavage via  upstream proteases, and it disrupts homeostatic processes and initiates an orderly disassembly of cells, including degradation of genomic DNA. 30The cleavage of PARP by caspase-3 is a well-characterized event in the apoptosis of animal cells. 31,32In the tetracaine-induced cell death, caspase-3 proform breakdown and the cleavage of PARP were detected after tetracaine treatment for 4 h. An 89-kd signature fragment of PARP was observed at 8 h (fig. 6).

PC12 cells were exposed to procaine, lidocaine, bupivacaine, or tetracaine for 10 h (table 1). Tetracaine at 1 mm induced cell death; 1 mm of other local anesthetics tested here did not influence cell survival. By increasing the concentration to 2 mm, three drugs except procaine induced different degrees of cell damage, and their relative potencies in the cell death assay followed the order tetracaine > bupivacaine > lidocaine.

Mitogen-activated protein kinase family phosphorylation in PC12 cells treated with 1 mm tetracaine was examined by Western blot analysis. ERK1 and ERK2 were phosphorylated 30 min after exposure to the drug. The ERK phosphorylation decreased after reaching a peak at 60 min (fig. 7A). Tetracaine also resulted in JNK1 and JNK2 phosphorylation within 30 min. After a greater decreasing at 2 h, both phosphorylated JNKs gradually increased again (fig. 7B). p38 phosphorylation was also induced within 30 min, and the maximal phosphorylation was observed at 30 min (fig. 7C).

PD98059, a MEK1 inhibitor, was used to explore which role ERKs play in the cell death and enhanced the tetracaine-induced cell death and LDH activity in the media (figs. 8A and B). Significantly larger numbers of dead cells were observed in the presence of 50 μm PD98059 than in its absence (P < 0.001). PD98059 with the absence of tetracaine was hardly detected to affect cell survival (data not shown).

PD98059 at 50 μm completely blocked the tetracine-induced ERK phosphorylation (fig. 8C). However, in the presence of PD98059, phosphorylation of JNK1 and JNK2 was enhanced at every time point (fig. 8D).

Tetracaine at lower concentrations (0.2 and 0.5 mm) also activated ERK phosphorylation, which took the same time course as the drug at higher concentration (1 mm;fig. 9A). Furthermore, the tetracaine-induced ERK activity occurred in a concentration-dependent manner (fig. 9A). PD98059 potently enhanced the effects of tetracaine at lower concentration on the PC12 cell survival (figs. 9B and C). The JNK phosphorylation could not be detected in the lower concentration treatment (data not shown). In the presence of 50 μm PD98059, 0.5 mm tetracaine induced a detectable JNK phosphorylation (fig. 9D), but 0.2 mm tetracaine did not (data not shown).

Curcumin, a dietary pigment in curry, has been indicated to be a potent inhibitor of JNK activation by interfering with the molecule(s) upstream of JNK in the signal transduction pathway, and it also inhibits ERK activation at a higher concentration. 33,34Curcumin at 10 μm partially inhibited the tetracaine-induced LDH activity released from damaged PC12 cells (fig. 10A) and the JNK phosphorylation (fig. 10B), but also slightly reduced the ERK phosphorylation (fig. 10C).

Tetracaine-treatment caused an increase in intracellular Ca²⁺concentration ([Ca²⁺]ⁱ) in PC12 cells, which nearly peaked at 15 min (fig. 11A). Both EGTA, a calcium-specific chelator, and BAPTA-AM, a chelator of intracellular calcium, significantly attenuated the action of tetracaine (fig. 11B). To understand the roles of increasing of [Ca²⁺]ⁱin tetracaine-induced apoptosis, the two calcium chelators were used. Tetracaine-induced LDH release from PC12 cells was diminished by minimizing extracellular Ca²⁺concentration with EGTA (fig. 12A). EGTA potentiated tetracaine-induced ERK phosphorylation and decreased JNK phosphorylation (figs. 12B and C). BAPTA-AM at 5 μm also reduced the tetracaine-induced cell damage (fig. 13A) and the JNK phosphorylation (fig. 13B). The tetracaine-induced ERK phosphorylation did not show dramatic increase (fig. 13C).

L-type Ca²⁺channels have been demonstrated to exist in PC12 cells. 27Thus, we examined role of the Ca²⁺channels in tetracaine-induced apoptosis. Nifedipine, a selective blocker of L-type Ca²⁺channels, neither induced the cell survival nor affected the action of tetracaine (table 2). Tetrodetoxin (0.5 mm) was introduced to examine whether blockade of Na⁺channel was involved in tetracaine-induced cell death, and we did not detect any effects of the Na⁺channel blocker on cell survival (table 2).

The major finding of the current study is that tetracaine, bupivacaine, and lidocaine, at their clinically relevant concentrations, induced PC12 cell death. The morphologic and biochemical changes associated with the tetracaine-induced cell death are characterized by plasma membrane bleb formation, chromatin condensation, nuclear fragmentation (or DNA ladder), and budding off of cellular fragments. 10Caspase-3 activation occurred in the process of the tetracaine-induced cell death. Phosphorylations of the MAPK family, ERK, JNK, and p38 were also observed in the process. Inhibition of ERK phosphorylation with a MEK1 inhibitor potentiated the tetracaine-induced JNK posphorylation and cell death. Curcumin, a JNK inhibitor, attenuated the cell damage. Although blocking of Na⁺channels was not likely involved in the action of tetracaine, EGTA, a Ca²⁺chelator, which per se  did not affect ERK and JNK phosphorylation, enhanced tetracaine-induced ERK phosphorylation and decreased JNK phosphorylation and prevented cell death. BAPTA-AM partially reduced tetracaine-induced cell death.

The morphologic changes of the cell surface are unique as the standpoint of apoptosis. Intramembranous particle distribution was markedly aggregated, and smooth intramembraneous particle-free areas appeared on the protoplasmic fracture in the presence of tetracaine. It is generally thought that the primary sites of local anesthetic action are at the cellular plasma membrane of the neuronal structures 35; tetracaine-induced morphologic changes are thus a very provocative finding. Because the time course for apoptotic cell death needed a longer time, it seems likely that tetracaine first triggers intracellular signaling pathways rather than its direct membrane effects, and the apoptosis-like morphologic changes are mediated by the intracellular signaling pathways including the MAPK family. On the process of such morphologic changes, movement of Ca²⁺from the exterior through the plasma membrane 36–38or from intracellular organella 39,40into the cytosol could play crucial roles, and increased [Ca²⁺]ⁱregulates several key steps in the apoptotic signaling pathway in various cell types, including neurons. 39,40Agents that suppress influx of Ca²⁺can prevent apoptosis in PC12 cells, 41such as unpermeable EGTA, which chelates extracellular Ca²⁺and abolishes influx of Ca²⁺. 38Local anesthetics such as lidocaine have been indicated to cause [Ca²⁺]ⁱincrease via  Ca²⁺influx through plasma membrane 2,7and to kill cells. 7In the current study, because EGTA abolished influx of Ca²⁺and kept the cells alive, an increase in [Ca²⁺]ⁱfrom an extracellular source is thought to play an important role in the tetracaine-induced apoptosis. BAPTA-AM, an esterified EGTA analog, goes into cytosol, where it is deesterified and binds Ca²⁺. Although local anesthetics were thought to block L-type Ca²⁺channels, 42and lidocaine at 30 mm would increase [Ca²⁺]ⁱvia  voltage-gated Ca²⁺channels, 2the effects of tetracaine seem not to be a result of its action on L-type Ca²⁺channels, because nifedipine neither induced apoptosis nor inhibited the tetracaine-induced cell death. We found that the role of the increased [Ca²⁺]ⁱvia  plasma membrane from the exterior is crucial in tetracaine-induced cell death, but one may not exclude other mechanisms such as membrane fluidity changes and alteration of mitochondrial functions, which have been reported to be involved in apoptosis. 43–45Indeed, it has been suggested that local anesthetics affect membrane fluidity 7and mitochondrial functions. 46,47 

To examine consequences of phosphorylation of ERK and JNK pathways in tetracaine-induced PC12 cell death, we used PD98059, an inhibitor of MEK1, which lies immediately upstream of ERK and is responsible for ERK phosphorylation or activation. 11PD98059 has been used to characterize the role of the ERK pathway in a lot of cell lines. 11,48,49For example, it has been reported that ERK activation protects neurons from glutamate toxicity, and PD98059 blocked the neuroprotection by inhibiting ERK activation. 50ERK activation is necessary for prevention of PC12 cell death by N -acetylcysteine, a promoter of neuronal survival, and PD98059 could completely inhibit the cell survival promotion. 51Thus, the current result indicating that inhibition of ERK phosphorylation by PD98059 increased tetracaine-induced cell death would suggest that ERK activation plays a protective role in the cellular survival pathway. Because EGTA pretreatment seems to protect PC12 cells from apoptosis by minimizing the toxic influx of Ca²⁺, enhancement of ERK phosphorylation by EGTA might suggest the recovery of ERK phosphorylation inhibited by the toxic influx of Ca²⁺(fig. 12). It is thus assumed that PC12 cells would activate ERK cascade to resist a potential action of tetracaine, but Ca²⁺influx induced by tetracaine per se  seems to overwhelm the protective cascade and then facilitate cell death.

It has been indicated that JNK cascade is involved in apoptosis in response to distinct stimuli in various cell types. 23Inhibition of c-Jun, the transcription factor activated by the JNK pathway, could protect the neurons from apoptosis, 52and increased activity of c-Jun seems to be sufficient to trigger apoptotic cell death in fibroblasts. 53Activation of JNK cascade has been reported to occur in the apoptosis induced by environmental stresses, ionizing radiation, hydrogen peroxide, ultraviolet C radiation, heat shock, tumor necrosis factor α, as well as PD98059. 54The current results indicating that curcumin inhibited tetracaine-induced JNK phosphorylation and cell death are in agreement with those of other reports. 33,34In addition to inhibiting ERK phosphorylation, PD98059 enhanced JNK phosphorylation and magnified tetracaine-induced cell death. The current findings may suggest that tetracaine-induced apoptotic cell death would occur as a result of losing balance of signaling molecular activities between survival signal, ERK cascade, and death signal, JNK cascade. Attenuation of JNK phosphorylation by EGTA and BAPTA-AM would suggest that overinflux of Ca²⁺might be upstream of JNK phosphorylation. p38 activation has been demonstrated not only to correlate with cell death, 12,55but also to play important roles in cell survival 48,56and the process of preconditioning. 57Thus, before investigating further in detail, we could not postulate which role p38 played in tetracaine-induced cell death.

In our previous studies, local anesthetics have been found to suppress important signal molecules, such as phospholipase D 58and muscarinic receptor-mediated ERK activation. 24ERK activation, which was found to be blocked with tetracaine (0.5 mm), lasted for 15 min (peaked at 2 min) after carbachol stimulation. 24The time course of KCl-stimulated ERK activation, which was completely blocked by tetracaine at 0.2 mm, just lasted for 10 min. 25In the current study, tetracaine-induced ERK phosphorylation was observed to last for several hours. Because of differences among these time courses, one can speculate that these pathways of ERK activation should differ. Tetracaine suppresses carbachol- or KCl-stimulated ERK activation by affecting the muscarinic receptor or L-type calcium channels. 24,25In addition, it remains to be elucidated whether tetracaine can direct act ERK per se  so far. It seems likely that tetracaine has different effects on distinct ERK pathways by diverse mechanisms. Thus, the activation of ERK cascade as a protective pathway may be a response of the cell itself to an irritant such as tetracaine by some unknown mechanism. Although the mechanism(s) for cell death other than apoptosis might be involved in PC12 cell death in the presence of tetracaine, we could exclude the possibility that necrotic cell death occurs in prolonged exposure of local anesthetics in any important way.

Local anesthetics used clinically rarely produce localized nerve damage. Reports of neurologic injury after single-dose and continuous spinal anesthesia suggest that clinically relevant concentrations of local anesthetics would cause neuronal injury. 5,6,59Although tetracaine at 0.2 mm scarcely induced cell death, ERK activation might indicate that cells responded to this concentration of the drug to active protective signal pathway to resistant extrairritant. It is well known that concentrations of three major clinical local anesthetics—lidocaine, bupivacaine, and tetracaine—for peripheral and central anesthesia are 1–5% (approximately 37–185 mm), 0.5–0.75% (approximately 15–23 mm), and 0.25–0.5% (approximately 8–17 mm), respectively. Because of vascular absorption by regional circulation and dilution by cerebrospinal fluid, their anesthetic concentrations are definitely lower than the used concentrations. During spinal anesthesia, the mean concentration of tetracaine in human cerebrospinal fluid has been reported to be approximately 0.02–0.4 mm. 60Because neuronal tissues would be exposed to local anesthetics at much higher concentrations than their anesthetic concentrations, it may be possible that apoptotic cell death around their injected sites could occur. Local anesthetics have been considerably safely used; however, prolonged use of local anesthetics for spinal or epidural anesthesia seems to be no longer simply safe and anesthesiologists should be wary of injecting them at extremely high concentrations in the vicinity of the neuronal structure, especially near the spinal cord. Because of significant differences between in vitro  observations and clinical observations, at clinically useful doses, effects of local anesthetics on neuronal structures might be different. Thus, more in vitro  and in vivo  studies are needed.

In summary, the current study indicates that tetracaine, bupivacaine, and lidocaine at millimole concentrations caused PC12 cell death, and tetracaine-induced cell death featured plasma membrane bleb formation, nuclear fragmentation, or condensation, chromatin fragmentation, and budding off of cellular fragments. Caspase-3 activation was observed in the tetracaine-induced cell death. Phosphorylation of the MAPK family, ERKs, JNKs, and p38 occurred in the process; ERK cascade and JNK cascade may play opposite roles to protect from or result in apoptosis (fig. 14). Toxic influx of Ca²⁺may damage cells by decreasing tetracaine-induced ERK phosphorylation and facilitating JNK phosphorylation. The action of local anesthetics on cell survival does not likely result from their effects on voltage-gated sodium channels and L-type calcium channels. The implications will help in understanding the mechanisms of the neurotoxicity of local anesthetics.

The authors thank Takashi Naganawa (Technician, Department of Biochemistry, Gifu University School of Medicine, Gifu City, Gifu, Japan) for technical assistance.