Kansha M, Nagata T, Irita K, Takahashi S
Department of Anesthesiology and Critical Care Medicine, Kysushu University School of Medicine, Fukuoka, Japan.
Anesthesiology. 1999 Dec;91(6):1798-806. doi: 10.1097/00000542-199912000-00034.
An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells.
The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting.
Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains.
Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.
细胞内钙水平的升高由N-甲基-D-天冬氨酸受体和L型Ca2+通道共同介导，可激活参与突触修饰的丝裂原活化蛋白（MAP）激酶信号通路。最近有研究表明，MAP激酶在将突触兴奋与突触后神经元细胞核中的基因表达偶联过程中发挥作用。由于局部麻醉药对神经元细胞中细胞信号转导的影响尚不清楚，因此作者使用PC12细胞研究了它们是否会影响MAP激酶信号通路。
用50 mM氯化钾或1 microM离子霉素刺激细胞，从而免疫沉淀活化的MAP激酶。然后对免疫复合物进行Elk1磷酸化测定。通过免疫印迹检测MAP激酶的磷酸化和c-Fos的诱导。
用1 mM（乙二氧基）-二乙二胺四乙酸或5微米硝苯地平预处理细胞可阻断50 mM氯化钾诱导的MAP激酶活化，而用2 microMω-芋螺毒素GIVA预处理则不能。丁卡因、丁哌卡因（抑制阳性对照活性50%的浓度[IC50s]分别为16.2±0.2和73.2±0.7 microM）以及丝裂原活化/细胞外受体调节激酶抑制剂PD 98059也可抑制氯化钾诱导的c-Fos表达。与氯化钾（丁卡因和丁哌卡因的IC50值分别为17.7±1.0和70.2±1.2 microM）相比，需要更高浓度的丁卡因和丁哌卡因才能抑制离子霉素诱导的MAP激酶活化（丁卡因和丁哌卡因的IC50值分别为62.5±2.2和330.5±32.8 microM）。尽管这些局部麻醉药的可能靶点可能是L型Ca2+通道或MAP激酶途径中Ca2+与Ras之间的成分，但它们直接影响MAP激酶的可能性仍然存在。
发现临床浓度的丁卡因和丁哌卡因可抑制PC12细胞中L型Ca2+通道介导的MAP激酶活化和c-Fos表达。因此，MAP激酶途径的抑制可能是丁卡因和丁哌卡因作用的潜在靶点，包括对突触功能的修饰。
