Dibucaine and tetracaine inhibit the activation of mitogen-activated protein kinase mediated by L-type calcium channels in PC12 cells

Background: 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. Methods: The cells were stimulated with either 50 mm KCl or 1 μM 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. Results: Pretreatment of the cells with 1 nM (ethylenedioxy)- diethyl-enedinitrilotetraacetic acid or 5 μ nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 μM ω- 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 μM, 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 μM, respectively) compared with potassium chloride (the IC₅₀ values of dibucaine and tetracaine were 17.7 ± 1.0 and 70.2 ± 1.2 μM, 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. Conclusions: 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.