Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death.
BACKGROUND
To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis.


METHODS
Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye.


RESULTS
Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Caspase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis.


CONCLUSIONS
Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaine's action for apoptosis.
Tan
Zhiming
Z
Departments of Anesthesiology and Critical Care Medicine.
Dohi
Shuji
S
Chen
Jinen
J
Banno
Yosiko
Y
Nozawa
Yoshinori
Y
D000779:Anesthetics, Local / Q000633:toxicity*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000648:ultrastructure; D003474:Curcumin / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005614:Freeze Fracturing; D048031:JNK Mitogen-Activated Protein Kinases; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D013748:Tetracaine / Q000633:toxicity*
D000779:Anesthetics, Local; D000970:Antineoplastic Agents; D002121:Calcium Channel Blockers; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D013748:Tetracaine; D007770:L-Lactate Dehydrogenase; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003474:Curcumin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium