Distinct Pharmacologic Properties of Neuromuscular Blocking Agents On

Anesthesiology 2006; 105:521–33 Copyright © 2006, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Distinct Pharmacologic Properties of Neuromuscular Blocking Agents on Human Neuronal Nicotinic Acetylcholine Receptors A Possible Explanation for the Train-of-four Fade Malin Jonsson, M.D., Ph.D.,* David Gurley, M.S.,† Michael Dabrowski, Ph.D.,‡ Olof Larsson, Ph.D.,§ Edwin C. Johnson, Ph.D.,# Lars I. Eriksson, M.D., Ph.D.࿣ Background: Nondepolarizing neuromuscular blocking agents intubation and mechanical ventilation and to improve Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/105/3/521/362271/0000542-200609000-00016.pdf by guest on 23 September 2021 (NMBAs) are extensively used in the practice of anesthesia and surgical conditions. intensive care medicine. Their primary site of action is at the Although it is well established that nondepolarizing postsynaptic nicotinic acetylcholine receptor (nAChR) in the ␣ ␤ ␧␦ neuromuscular junction, but their action on neuronal nAChRs NMBAs block the postsynaptic 1 1 nicotinic acetyl- have not been fully evaluated. Furthermore, observed adverse choline receptor (nAChR) subtype at the muscle end- effects of nondepolarizing NMBAs might originate from an in- plate, the effect on the presynaptic motor nerve ending teraction with neuronal nAChRs. The aim of this study was to has not been clarified (for a review, see Vizi and Lend- examine the effect of clinically used nondepolarizing NMBAs on 1,2 2 muscle and neuronal nAChR subtypes. vai and Bowman et al. ). It is believed that the mech- Methods: Xenopus laevis oocytes were injected with messen- anism behind tetanic and train-of-four (TOF) fade during ger RNA encoding for the subunits included in the human neuromuscular block by a nondepolarizing NMBA arise ␣ ␤ ␧␦ ␣ ␤ ␣ ␤ ␣ ␤ ␣ 1 1 , 3 2, 3 4, 4 2, and 7 nAChR subtypes. The interac- from an interaction with presynaptic cholinergic autore- tions between each of these nAChR subtypes and atracurium, ceptors at the motor nerve ending.1,3 However, the cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium were studied using an eight-chan- affinity of nondepolarizing NMBAs to such presynaptic nel two-electrode voltage clamp setup. Responses were mea- autoreceptors has not been investigated at the molecular sured as peak current and net charge. level. Further, it has recently been shown that an inhi- Results: All nondepolarizing NMBAs inhibited both muscle ␣ ␤ bition of the presynaptic 3 2 nAChR subtype at the and neuronal nAChRs. The neuronal nAChRs were reversibly motor nerve end4 induces tetanic fade.5 Based on this, it and concentration-dependently inhibited in the low micromo- lar range. The mechanism (i.e., competitive vs. noncompetitive) seems likely that the tetanic fade phenomenon seen of the block at the neuronal nAChRs was dependent both on during nondepolarizing neuromuscular block is due to ␣ ␤ subtype and the NMBA tested. The authors did not observe an inhibition of the 3 2 nAChR subtype. activation of the nAChR subtypes by any of the NMBAs tested. ␣ ␤ ␧␦ ␣ ␤ The 1 1 and the 3 2 nAChRs are members of the Conclusions: The authors conclude that nondepolarizing same neurotransmitter-gated ion channel superfamily. NMBAs concentration-dependently inhibit human neuronal ␣ ␤ They are composed of five transmembrane subunits with nAChRs. The inhibition of the presynaptic 3 2 nAChR subtype expressed at the motor nerve ending provides a possible mo- a central cation pore, and the stoichiometry and identity lecular explanation for the tetanic and train-of-four fade seen of subunits determines each receptor’s unique proper- during a nondepolarizing neuromuscular block. ties.6 To date, 17 nicotinic subunits have been cloned in ␣ ␤ ␦ ␥ ␧ vertebrates: the muscle 1, 1, , , and subunits and ␣ ␤ 7 NONDEPOLARIZING neuromuscular blocking agents the neuronal 2–10 and 2–4 subunits. Although there (NMBAs) are extensively used in the practice of anesthe- are many potential combinations of neuronal nAChRs, sia and intensive care medicine to facilitate tracheal only a few have as yet been found to be of biologic importance.8,9 The neuronal nAChRs are found both * Resident, ࿣ Professor, Department of Anesthesiology and Intensive Care presynaptically and postsynaptically in neurons of the Medicine, Karolinska University Hospital and Karolinska Institutet. † Principal ␣ ␤ ␣ ␤ ␣ 9,10 central ( 4 2, 3 2, 7) and peripheral nervous sys- Scientist, Director, Target Biology, AstraZeneca Pharmaceuticals, Wilmington, ␣ ␤ ␣ ␤ ␣ 9,11,12 Delaware. ‡ Senior Research Scientist, § Associate Professor, Molecular Phar- tem ( 3 4, 3 2, 7) as well as in extraneuronal macology, AstraZeneca R&D, So¨derta¨lje, Sweden. tissues and cells, such as keratinocytes, muscle, lympho- Received from the Department of Anesthesiology and Intensive Care Medi- cytes, macrophages, carotid bodies, and neurosecretory cine, Karolinska University Hospital, Stockholm, and Karolinska Institutet, Stock- 6,7,13 holm, Sweden. Submitted for publication February 15, 2006. Accepted for pub- cells. lication May 5, 2006. Supported by grants from the Swedish Research Council Interactions between NMBAs and neuronal nAChRs (project No. 10401), Olof Norlander Travel Grant, the Stockholm County Coun- cil, the Swedish Society of Medicine, Karolinska Institutet Travel Grants and may cause serious cardiovascular and respiratory side Funds, and Hirsch Fellowship, all Stockholm, Sweden. GlaxoSmithKline, Barnard effects. It has been shown that nondepolarizing NMBAs Castle Durham, United Kingdom, provided atracurium and cisatracurium; NV Organon, BH Oss, The Netherlands, provided pancuronium, rocuronium, and reduce hypoxic ventilatory response in partially para- vecuronium; and AstraZeneca Pharmaceuticals, Wilmington, Delaware, provided lyzed humans,14,15 and the mechanism behind this de- messenger RNA for the nicotinic acetylcholine receptor subunits. Address correspondence to Dr. Jonsson: Department of Anesthesiology and pression might be interference with nicotinic chemo- 16,17 Intensive Care Medicine, and Karolinska Institutet, Karolinska University Hospi- transduction of the carotid bodies. At the molecular tal, Stockholm, SE-171 76 Stockholm, Sweden. [email protected]. In- dividual article reprints may be purchased through the Journal Web site, level, d-tubocurarine, pancuronium, atracurium, and its www.anesthesiology.org. degradation product laudanosine have been shown to Anesthesiology, V 105, No 3, Sep 2006 521 522 JONSSON ET AL. block neuronal nAChR subtypes expressed in Xenopus lated by partial ovariectomy from frogs anesthetized oocytes.18–22 Interestingly, some reports indicate that with 0.2% Tricaine (Sigma, St. Louis, MO). The ovaries ␣ ␤ ␥␦ ␣ ␤ NMBAs can act as partial agonists at 1 1 , 3 4, and were mechanically dissected to smaller lumps and di- ␣ ␤ 21,23 4 2 nAChR subtypes ; however, other studies could gested in OR-2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM 20,24 not demonstrate any agonism by NMBAs. MgCl2,5mM HEPES, pH adjusted to 7.5 with NaOH) ␣ The 7 nAChR subtype plays a key role in the cholin- containing 1.5 mg/ml collagenase (type 1A; Sigma) for ergic reflex involved in inflammatory conditions such as 90 min to remove the follicular epithelia from the oo- sepsis,25,26 and it can be speculated whether NMBAs cytes. After 1–24 h, the oocytes were injected with used in intensive care settings might interact with the 0.2–18 ng mRNA in a total volume of 30–40 nl/oocyte. inflammatory response to sepsis. Furthermore, although Multiple subunit combinations were injected at a 1:1 ␣ ␤ ␧␦ ␣ ␤ ␣ ␤ highly charged, NMBAs can under certain conditions ratio ( 1 1 or x y), except for 4 2, where the in- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/105/3/521/362271/0000542-200609000-00016.pdf by guest on 23 September 2021 cross the blood–brain barrier,27–29 thus having the po- jection ratio was 1:9. The oocytes were maintained in tential to interact with central cholinergic receptors and Leibovitz L-15 medium (Sigma) diluted 1:1 with Millipore 30 31,32 ␮ the synaptic transmission and cause seizures. filtered double distilled H2O (Billerica, MA) and 80 g/ml Because most nondepolarizing NMBAs were devel- gentamicin, 100 U/ml penicillin, and 100 ␮g/ml strepto- oped before cloning and isolation of their target pro- mycin added. Oocytes were incubated at 18°–19°C for teins, the precise modes of action have not been exam- 2–7 days after injection before being studied. ined in detail. A better understanding of the molecular mechanisms of action of clinically used nondepolarizing Electrophysiologic Recordings NMBAs on human neuronal nAChR subtypes is needed. All recordings were performed at room temperature In addition, for future drug design, it is essential to define (20°–22°C). During recording, the oocytes were contin- potential interactions with human neuronal nAChRs. uously perfused with ND-96 (96.0 mM NaCl, 2.0 mM KCl, The aim of this study was therefore to investigate the 1.8 mM CaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, pH 7.4 potency and functional affinity of clinically used nonde- adjusted with NaOH). Oocyte recordings were per- polarizing NMBAs on acetylcholine-induced responses formed using an integrated system that provides auto- on human muscle and neuronal nAChRs heterologously mated impalement of up to eight oocytes, studied in expressed in Xenopus oocytes. In addition, potential parallel with two-electrode voltage clamp, and current activation of nAChRs by nondepolarizing

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