Influence of Quantal Transmitter Release and Nicotinic Acetylcholine Receptors

Background: The diaphragm is resistant to competitive neuromuscular blocking agents, as compared to peripheral muscles. The basis of this difference may be a higher concentration of acetylcholine released or higher number of postsynaptic nicotinic acetylcholine receptors in diaphragmatic neuromuscular junctions. Methods: Nerve-evoked twitch-tension was measured in rat hemidiapragm as was Extensor digitorum longus (EDL) nervemuscle preparation to determine the effective D-tubocurarine concentration that decreased twitch responses by 50%. The mean quantal content of endplate potentials was determined in single junctions in a low-Ca , high-Mg Krebs-Ringer medium. Strips of hemidiaphragm and EDL muscle, containing the endplate regions, were used to determine the number of nAChR nicotinic acetylcholine receptor binding sites with the aid of radiolabeled [I] -bungarotoxin. Results: The effective D-tubocurarine concentration that decreased twitch responses by 50% (median [interquartile range]) was seven-fold higher in the hemidiaphragm than in the EDL (1.82 M [1.43–2.20] vs. 0.26 M [0.23–0.29], P < 0.01). The median of the mean quantal content was higher in the hemidiaphragm than in the EDL (0.57 [0.44–0.84] vs. (0.14 [0.11–0.19], P < 0.01). The number of specific [I] -bungarotoxin binding sites to junctional nicotinic acetylcholine receptors was higher in the diaphragm than in the EDL (1.15 fmol/mg [0.48–1.70] vs. 0.55 fmol/mg [0.23–0.70 ] , P < 0.05). Conclusion: The current study indicates that the resistance of the diaphragm to neuromuscular blocking agents can be explained by both a higher mean quantal content of endplate potentials and a higher number of nicotinic acetylcholine receptor binding sites than in the peripheral EDL muscle. THE diaphragm is resistant to the blocking effect of competitive neuromuscular blocking agents (NMBA), as compared to peripheral muscles. Dose-response curves demonstrated a shift to the right of the diaphragmatic response compared to the Adductor pollicis muscle in humans. After an intubating dose of a competitive NMBA in anesthetized patients, the recovery of the diaphragm-evoked response occurs earlier than at the Adductor pollicis muscle. The resistance of the diaphragm to NMBA is still poorly understood. Muscle type composition, which differs between the diaphragm and peripheral muscles, does not explain the difference in muscle relaxant effect. In the cat, Waud and Waud demonstrated that the safety margin of neuromuscular transmission in the diaphragm was greater than in the Tibialis anterior muscle, but they could not provide any explanation for this difference. The mechanism of resistance may be either presynaptic or postsynaptic. Presynaptic factors include the modulation of acetylcholine release from motor nerve terminals. Postsynaptic factors include the density of nicotinic acetylcholine receptors (nAChR) and the rate of acetylcholine hydrolysis by acetylcholinesterase. We recently measured acetylcholinesterase activity of the different heterooligomers of the neuromuscular junction in the diaphragm and in a peripheral mouse limb muscle. Although acetylcholinesterase activity was lower in the diaphragm than in the Extensor digitorum longus (EDL), this difference could not explain the diaphragmatic resistance to tubocurarine because specific inhibition of acetylcholinesterase did not change the four-fold effective D-tubocurarine dose-ratio between the diaphragm and the EDL observed in the mouse. In the current study, we investigated whether the diaphragmatic resistance to D-tubocurarine depends on the quantal content of endplate potentials and/or on the number of nAChR binding sites in the neuromuscular junctions of the diaphragm and the EDL.