Anesthesiology 2009; 110:1011–5 Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Resistance to D-Tubocurarine of the Rat Diaphragm as Compared to a Limb Muscle Influence of Quantal Transmitter Release and Nicotinic Acetylcholine Receptors Tu Nguyen-Huu, M.D.,* Jordi Molgo ´ , D.D.S., Ph.D., D.Sci.,† Denis Servent, Ph.D.,‡ Philippe Duvaldestin, Ph.D. M.D.§ Background: The diaphragm is resistant to competitive neu- romuscular blocking agents, as compared to peripheral mus- cles. The basis of this difference may be a higher concentration of acetylcholine released or higher number of postsynaptic nicotinic acetylcholine receptors in diaphragmatic neuromus- cular junctions. Methods: Nerve-evoked twitch-tension was measured in rat hemidiapragm as was Extensor digitorum longus (EDL) nerve- muscle 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 2 , high-Mg 2 Krebs-Ringer me- dium. 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 [ 125 I]-bungarotoxin. Results: The effective D-tubocurarine concentration that de- creased 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 hemidi- aphragm than in the EDL (0.57 [0.44 – 0.84] vs. (0.14 [0.11– 0.19], P < 0.01). The number of specific [ 125 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 ex- plained by both a higher mean quantal content of endplate potentials and a higher number of nicotinic acetylcholine re- ceptor 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. 1–3 After an intubating dose of a competitive NMBA in anesthetized patients, the recovery of the dia- phragm-evoked response occurs earlier than at the Ad- ductor pollicis muscle. 4–7 The resistance of the dia- phragm 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. 8–9 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. 10 The mechanism of resistance may be either presynap- tic or postsynaptic. Presynaptic factors include the mod- ulation of acetylcholine release from motor nerve termi- nals. Postsynaptic factors include the density of nicotinic acetylcholine receptors (nAChR) and the rate of acetyl- choline 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. 11 Although acetylcholinesterase activity was lower in the diaphragm than in the Extensor digitorum longus (EDL), this difference could not explain the diaphrag- matic resistance to tubocurarine because specific inhibi- tion of acetylcholinesterase did not change the four-fold effective D-tubocurarine dose-ratio between the diaphragm and the EDL observed in the mouse. 11 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. Materials and Methods Animals The study was approved by the Animal Ethics Commit- tee of the Centre National de la Recherche Scientifique. All experiments were performed in accordance with European Community guidelines for animal laboratory handling. This study, including care of animals, was conducted according to the official edict presented by * Assistant Professor of Anesthesia, Centre National de la Recherche Scienti- fique, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neu- robiologie Cellulaire et Mole ´culaire – UPR9040, 91198 Gif sur Yvette Cedex, France, and Service d’Anesthe ´sie-Re ´animation, Ho ˆpital Henri Mondor, Assistance Publique-Ho ˆpitaux de Paris, Cre ´teil, France; † Research Director, Centre National de la Recherche Scientifique, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie Cellulaire et Mole ´culaire – UPR9040, 91198 Gif sur Yvette Cedex, France; ‡ Research Director, Commissariat a ` l’Energie Atomique, Institut de biologie et de technologies de Saclay, Service d’Inge ´nierie Mole ´culaire des Prote ´ines, Laboratoire de Toxinologie Mole ´culaire, 91191 Gif sur Yvette, France; § Professor, Service d’Anesthe ´sie-Re ´animation, Ho ˆpital Henri Mondor, Assistance Publique-Ho ˆpitaux de Paris, Cre ´teil, France. Received from Laboratoire de Neurobiologie Cellulaire et Mole ´culaire, Unite ´ Propre de Recherche 9040, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France, and Service d’Anesthe ´sie-Re ´animation, Ho ˆpital Henri Mon- dor, Assistance Publique-Ho ˆpitaux de Paris, Cre ´teil, France. Submitted for publication April 21, 2008. Accepted for publication January 8, 2009. Sup- ported in part by the Centre National de la Recherche Scientifique, Gif sur Yvette, France, the Association Franc ¸aise contre les Myopathies, Evry, France (to Dr. Molgo ´), and the Commissariat a ` l’Energie Atomique, Saclay, France (to Dr. Servent). Dr. Nguyen-Huu’s current position: Professor of Anesthesia, Hanoi Medical University, and Department of Anesthesiology, Viet Duc Hos- pital, Hanoi, Vietnam. Address correspondence to Dr. Duvaldestin: Service d’Anesthe ´sie-Re ´anima- tion, Ho ˆpital Henri Mondor, Assistance Publique-Ho ˆpitaux de Paris, Cre ´teil, France. philippe.duvaldestin@hmn.aphp.fr. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. Anesthesiology, V 110, No 5, May 2009 1011 Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/931085/ on 10/15/2017