Cholinesterases and the resistance of the mouse diaphragm to the effect of tubocurarine

Background. The diaphragm is resistant to competitive neuromuscular blocking agents. Because of the competitive mechanism of action of tubocurarine, the rate of hydrolysis of acetylcholine at the neuromuscular junction may modulate its neuromuscular blocking effect. The authors compared the neuromuscular blocking effect of tubocurarine on isolated diaphragm and extensor digitorum longus (EDL) muscles and quantified the acetylcholinesterase activity in hetero-oligomers. Methods: Adult Swiss-Webster and collagen Q-deficient (ColQ(-/-)) mice were used. The blocking effect of tubocurarine on nerve-evoked muscle twitches was determined in isolated diaphragm and EDL muscles, after inhibition of acetylcholinesterase by fasciculin-1, butyrylcholinesterase by tetraisopropylpyrophosphoramide, or both acetylcholinesterase and butyrylcholinesterase by neostigmine, and in acetylcholinesterase-deficient ColQ(-'-) muscles. The different acetylcholinesterase oligomers extracted from diaphragm and EDL muscles were quantified in sucrose gradient. Results: The EC50 for tubocurarine to decrease the nerve-evoked twitch response was four times higher in the diaphragm than in the EDL. The activity of the different acetylchohnesterase oligomers was lower in the diaphragm as compared with the EDL. Inhibition of acetylcholinesterase by antagonists resulted in an increased dose of tubocurarine but an unchanged resistance ratio between the diaphragm and the EDL. A similar diaphragmatic resistance was found in ColQ-/- muscles. Conclusion: The current study indicates that, despite differences in acetylcholinesterase activity between the diaphragm and EDL, the diaphragmatic resistance to tubocurarine cannot be explained by the different rate of acetylcholine hydrolysis in the synaptic cleft.