Local anesthetics inhibit muscarinic receptor-mediated activation of extracellular signal-regulated kinases in rat pheochromocytoma PC12 cells.
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 4beta-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.
Tan
Z
Z
Department of Anesthesiology and Critical Care Medicine, Gifu University School of Medicine, Gifu City, Japan.
Dohi
S
S
Ohguchi
K
K
Nakashima
S
S
Nozawa
Y
Y
D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011343:Procaine / Q000494:pharmacology*; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D000779:Anesthetics, Local; D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D014443:Tyrosine; D013779:Tetrodotoxin; D011343:Procaine; D002217:Carbachol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate