Local anesthetics inhibit muscarinic receptor-mediated activation of extracellular signal-regulated kinases in rat pheochromocytoma PC12 cells

Abstract

Background: Because protein phosphorylation is a key mechanism for controlling cellular functions and extracellular signal-regulated kinase (ERK) plays a role in cellular signal transduction, the authors wanted to determine whether local anesthetics interfere with biochemical signaling molecules. Methods: Protein tyrosine phosphorylation and ERK activation induced by carbachol, an agonist for muscarinic acetylcholine receptors, were examined in rat pheochromocytoma PC12 cells, a model for investigating signal transduction. Carbachol-induced tyrosine-phosphorylated proteins of 44 and 42 kd were determined by Western blot analysis and identified as activated ERK1 and ERK2 using anti-ERK antibody. The ERK activation was blocked by preincubation with atropine or an M3 muscarinic acetylcholine receptor antagonist 4-diphenyacetooxy-1, 1-dimethylpiperidinium, indicating that is was mediated by M3 muscarinic acetylcholine receptor activation. Then, in the presence of local anesthetic, the carbachol-induced tyrosine phosphorylation and ERK activation were evaluated. The effects of three Na+ current-modifying reagents on carbachol-induced ERK activation were also evaluated. Results: Procaine (10-4 to 10-3 M) inhibited carbachol-induced tyrosine phosphorylation and ERK activation in a concentration-dependent manner. Although tetracaine, lidocaine, and bupivacaine similarly suppressed carbachol-induced tyrosine phosphorylation and ERK activation, neither tetrodotoxin, veratridine, nor ouabain affected the carbachol induced ERKs activation. Both ERKs were also activated by 4β phorbol 12-myristate 13- acetate, an activator of protein kinase C, and fluoroaluminate (AlF4/-), respectively, but procaine did not affect ERK activation induced by these two substances. The inhibition of carbachol-induced ERK activation by procaine was not modified by a phosphatase inhibitor, calyculin A. Conclusions: The current results indicate that local anesthetics inhibit the activity of the signal-transducing molecule(s) leading to M3 muscarinic acetylcholine receptor-mediated ERK activation in PC12 cells. Such action is unlikely to be a result of the drug's action on Na+ channels or on the electrochemical gradients of the neuronal cell membrane.