Lack in Effects of Therapeutic Concentrations of Dexmedetomidine and Clonidine on the Neuromuscular

Lack in Effects of Therapeutic Concentrations of Dexmedetomidine and Clonidine on the Neuromuscular Blocking Action of Rocuronium in Isolated Rat DiaphragmsEichi Narimatsu,MD,PhD*†Tomohisa Niiya,MD,PhD* Mikito Kawamata,MD,PhD* Akiyoshi Namiki,MD,PhD*BACKGROUND:We investigated the effects of the␣-2adrenoceptor agonists clonidine and dexmedetomidine on the neuromuscular blocking effect of rocuronium in vitro. METHODS:Isometric twitch tensions of rat nerve–hemidiaphragm preparations elicited by indirect(phrenic nerve)supramaximal stimulation at0.1Hz were evaluated.RESULTS:Clonidine and dexmedetomidine50␮M(nϭ6each),but not0.05␮M, shifted the rocuronium concentration–twitch tension curves to the left and de-creased the rocuronium concentration for50%twitch depression(IC50)compared with control(nϭ9,PϽ0.01).The leftward shift induced by clonidine50␮M or dexmedetomidine50␮M was not antagonized by yohimbine50␮M,an␣-2 adrenoceptor antagonist.Twitch tensions partially depressed by7␮M rocuronium, to about65%of the control,were further suppressed in a concentration-dependent manner by clonidine(nϭ6)and dexmedetomidine(nϭ9)at concentrations of30␮M or more(PϽ0.01).CONCLUSIONS:These results indicate that very high,but not therapeutic,concentra-tions of clonidine and dexmedetomidine enhance the neuromuscular blocking action of rocuronium,but this is not mediated by an agonist action on␣-2 adrenoceptors.(Anesth Analg2007;104:1116–20)C oncurrently administered drugs could affect theactions of nondepolarizing neuromuscular blocking drugs.Clonidine and dexmedetomidine,␣-2adrener-gic agonists,have been used in the perioperative period and in intensive care for sedation,analgesia, and management of hypertension.However,the ef-fects of␣-2adrenergic agonists on the actions of nondepolarizing neuromuscular blocking drugs have not been extensively studied.The in vivo effects of␣-2adrenergic agonists on the actions of nondepolarizing neuromuscular blocking drugs are conflicting.Both enhancement(1)and no significant alteration(2)have been reported for the effect of oral clonidine on vecuronium-induced muscle relax-ation.Dexmedetomidine enhances rocuronium-induced muscle relaxation in volunteers(3)but does not signifi-cantly alter vecuronium-induced muscle relaxation in rats in vivo(4).In vitro studies have revealed that clonidine itself depresses neuromuscular transmission but that its␣-2 agonist effect may not contribute to the depression. Clonidine does not significantly alter acetylcholine re-lease from motor nerve terminals(5).Clonidine de-presses the endplate potential by blocking postjunctional nicotinic acetylcholine receptors,but not by acting on␣-2 adrenoceptors(6,7).Clonidine does not significantly alter d-tubocurarine-induced partial twitch depression in vitro(8).The in vitro effects of dexmedetomidine on the actions of nondepolarizing neuromuscular blocking drugs have not been investigated.This study was designed to directly test the hypoth-esis that␣-2adrenergic agonists pharmacodynamically enhance the actions of nondepolarizing neuromuscular blocking drugs.We investigated the in vitro effects of clinical and experimental concentrations of clonidine and dexmedetomidine on the neuromuscular blocking action of rocuronium.METHODSThe study was approved by the Animal Care and Use Committee of Sapporo Medical University.Adult male Wistar rats(7–9Wk old and weighing235–270g,From the Departments of*Anesthesiology and†Traumatology and Critical Care Medicine,Sapporo Medical University School of Medicine,Sapporo,Japan.Accepted for publication January11,2007.Address correspondence and reprint requests to Dr.Eichi Narimatsu,MD,PhD,Department of Anesthesiology,Sapporo Medical University School of Medicine,South1,West16,Chuo-ku, Sapporo060-8543,Japan.Address e-mail to enarimat@sapmed.ac.jp.Copyright©2007International Anesthesia Research Society DOI:10.1213/01.ane.0000260317.02748.83nϭ84)were killed by an overdose of isoflurane in oxygen and exsanguinated.Left hemidiaphragms with attached phrenic nerve,central tendon,and intact ribcage were rapidly removed.Strips of dia-phragm(10mm in width)with the phrenic nerve attached,which had been cut parallel to the muscle fibers,were then dissected.Each isolated strip was mounted vertically in a tissue chamber(25mL in volume),fixed at the ribcage,inferiorly positioned and suspended at the central tendon from a force displace-ment transducer(FT-03,Grass,Quincy,MA)using a 3-0silk suture.The chamber was filled with modified Kreb’s solution maintained at27°C and bubbled with 95%O2–5%CO2.The composition of the modifiedKreb’s solution was(in millimolar)NaCl,118.0;KCl,3.7;CaCl2,2.5;MgCl2,1.3;NaHCO3,26.2;Na2HPO4,1.2;and glucose,11;and pH was7.40Ϯ0.05whilebubbling.Twitch tension was elicited by indirect(phrenicnerve)or direct(muscle)supramaximal constant cur-rent stimulation at0.1Hz(for0.05and for0.2ms inthe cases of indirect stimulation and direct stimula-tion,respectively)using a stimulator(S48,Grass)anda constant current unit with an optimal preload(2.5–5.0g)to deliver maximal twitch tension.Forindirect stimulation,the phrenic nerve was positionedon wire bipolar platinum electrodes and electricallystimulated.For direct stimulation,a diaphragm speci-men,pretreated with a sufficient concentration(12␮M)of d-tubocurarine to completely abolish neuro-muscular transmission,was positioned between a pairof plate bipolar platinum electrodes(each25mmϫ12mm)and electrically stimulated.The twitch tensionwas recorded via the force transducer on a thermalchart recorder.After the twitch tension had been stabilized for atleast30min,single twitch tension(averaged in groupsof five)was measured and taken as a control.A studydrug was then added to the bathing solution.Afterstabilization of the drug effect for at least10min,twitch tension was again measured.The drug concen-trations were obtained by adding a freshly preparedsolution(dissolved in modified Kreb’s solution)ofrocuronium(100␮M or10mM),dexmedetomidine(5 or400␮M),clonidine(5␮M or5mM),or yohimbine (5mM)with calibrated micropipettes into25mL of modified Kreb’s solution in the tissue chamber.Rela-tionships between rocuronium concentration and indirectly elicited twitch tension were investigated using control diaphragm strips with no pretreatment and those pretreated with dexmedetomidine(0.05or 50␮M),clonidine(0.05or50␮M),yohimbine(50␮M), dexmedetomidineϩyohimbine(50␮M each),or clonidineϩyohimbine(50␮M each)(Table1).To investigate the effects of dexmedetomidine and clonidine on rocuronium-induced twitch depression, dexmedetomidine(nϭ9),clonidine(nϭ6),or a sham drug(traces of modified Kreb’s solution,nϭ6)was applied as supplement and in a stepwise manner after the7␮M rocuronium-induced twitch depression had stabilized.The effects of clonidine,dexmedetomidine, and yohimbine on indirectly or directly elicited twitch tension(without rocuronium)were also investigated by applying50␮M of one of these drugs(nϭ6each). Different rats were used in each experiment.Data were accepted only when twitch tension returned to 95%–105%of the initial value by rinsing the dia-phragm preparation with modified Kreb’s solution (indirect stimulation)or that containing12␮M d-tubocurarine(direct stimulation)in each study.The isometric twitch tension was measured by researchers who had not been informed of which experimental group each specimen belonged to.Rocuronium bro-mide was obtained from anon(Oss,The Netherlands).Dexmedetomidine hydrochloride was obtained from Abbott Laboratories(Abbott Park,IL). Clonidine hydrochloride,yohimbine hydrochloride(an ␣-2adrenergic antagonist),d-tubocurarine(a nondepo-larizing neuromuscular blocking drug),and all other drugs were purchased from Sigma(St.Louis,MO).Twitch tension data(%of the control value)are expressed as meansϮpetition analysis data were determined from a four-parameter logistic sigmoi-dal dose–response model fitted to the concentration–indirectly elicited twitch tension curves(All values were considered for analysis and twitch tension in the same preparation without rocuronium was defined as the control value)using the computer program Prism4 (GraphPad Software,San Diego,CA).Data of the50% inhibitory concentrations of the curves(IC50,in micro-molar);i.e.,the rocuronium concentration for50%twitch depression,are expressed as means with95%confidence intervals.Data of log IC50(in log␮M)and slope at log IC50are expressed as meansϮse.Statistical significance in IC50s was calculated from log IC50.One-or two-way repeated-measures analysis of variance(ANOVA) with post hoc(Scheffe’s F)testing and one-way factorial ANOVA with Scheffe´’s F test were used for statistical comparison,and PϽ0.05was accepted as significant.A shift of a curve was regarded as significant when two-way repeated-measures ANOVA demonstrated a statis-tically significant difference between the corresponding curves.Table1.Pretreatments Preceding Investigations of Rocuronium Concentration-Indirectly Elicited Twitch Tension RelationshipsPretreatmentConcentration(␮M)Number Control(no pretreatment)09 Dexmedetomidine0.056 Dexmedetomidine506 Clonidine0.056 Clonidine506 Yohimbine506 DexmedetomidineϩYohimbineeach506 ClonidineϩYohimbine each506RESULTSThere was no significant difference in body weights of rats and sizes of diaphragm preparations (width and length)in all of the experimental groups.There was also no significant difference in control twitch tensions elicited by indirect or direct stimulation (15.9–19.3g)among the experimental groups.Neither dexmedetomidine,clonidine nor yohimbine (each 50␮M)significantly altered the indirectly elicited twitch tensions and directly elicited twitch tensions.The effects of ␣-2agonists and an ␣-2antagonist on rocuronium-induced twitch depressions were investi-gated.Rocuronium reduced the indirectly elicited twitch tensions in the control and all pretreated dia-phragms in a concentration-dependent manner (P Ͻ0.01,Fig.1).There was no significant difference be-tween the curves or between the IC 50values in the control diaphragm and that pretreated with 0.05␮M dexmedetomidine or 0.05␮M clonidine (Fig.1and Table 2).Pretreatment of the diaphragm with 50␮M clonidine,50␮M clonidine ϩ50␮M yohimbine or 50␮M yohimbine significantly shifted the curve leftward from the curve for the control diaphragm (P Ͻ0.01,Fig.1A)and decreased IC 50s of those curves from that for the control diaphragm (P Ͻ0.01,Table 2).Similar to the action of 50␮M clonidine,pretreatment of the diaphragm with 50␮M dexmedetomidine or with 50␮M dexmedetomidine ϩ50␮M yohimbine signifi-cantly shifted the curve leftward from the curve for the control diaphragm (P Ͻ0.01,Fig.1B)and de-creased IC 50s of those curves from that for the control diaphragm (P Ͻ0.01,Table 2).The curve and its IC 50for the diaphragm pretreated with 50␮M dexmedeto-midine ϩ50␮M yohimbine significantly shifted left-ward from and was smaller than those for the diaphragm pretreated with 50␮M dexmedetomidine alone,respectively (P Ͻ0.01,Fig.1B and Table 2).There was no significant difference between the slopes at IC 50s of the curves for any of the control and pretreated diaphragms.The effects of dexmedetomidine and clonidine on rocuronium-induced partial neuromuscular block were investigated.Applications of rocuronium and dexmedetomidine or clonidine significantly depressed the indirectly elicited twitch tensions (P Ͻ0.01,Fig.2).The twitch tensions decreased by 7␮M rocuronium,to about 65%of the control,were further decreased in a concentration-dependent manner by dexmedetomi-dine or clonidine,and these significant decreases were observed at concentrations of 30␮M or more (PϽFigure 1.Concentration–twitch tension curves for rocuro-nium of isolated rat diaphragms pretreated with clonidine (A),dexmedetomidine (B)and/or yohimbine.Data are expressed as mean Ϯsd of nine and six experiments for control and pretreated diaphragms,respectively.Table 2.The 50%Inhibitory Concentrations (IC 50)and Slopes of Rocuronium Concentration–Twitch Tension Curves for Diaphragms Pretreated with Dexmedetomidine,Clonidine and/or YohimbineVariable Control Dexmedetomidine0.05␮M Clonidine 0.05␮M Yohimbine 50␮M Dexmedetomidine50␮M Clonidine 50␮M Dexmedetomidine50␮M ϩYohimbine 50␮M Clonidine 50␮M ϩYohimbine 50␮M log IC 50(log ␮M)Ϫ5.095Ϯ0.008Ϫ5.083Ϯ0.006Ϫ5.063Ϯ0.007Ϫ5.225Ϯ0.005*Ϫ5.292Ϯ0.006*Ϫ5.585Ϯ0.012*Ϫ5.697Ϯ0.004*†Ϫ5.712Ϯ0.009*IC 50(␮M)8.0388.2518.659 5.952 5.102 2.602 2.011 1.941(7.763–8.323)(8.021–8.488)(8.390–8.938)(5.822–6.084)(4.967–5.241)(2.465–2.747)(1.977–2.046)(1.864–2.020)Slope at IC 50Ϫ4.226Ϯ0.254Ϫ4.641Ϯ0.242Ϫ4.217Ϯ0.230Ϫ5.421Ϯ0.301Ϫ4.740Ϯ0.325Ϫ4.799Ϯ0.428Ϫ5.511Ϯ0.761Ϫ5.747Ϯ1.756Number96666666IC 50ϭ50%inhibitory concentration of the rocuronium concentration-twitch tension curve.Values are expressed as mean with 95%confidence interval in IC 50(␮M)and mean ϮSE in log IC 50(log ␮M)and slope at log IC 50.Statistical significance in IC 50was calculated from log IC 50.*P Ͻ0.01vs.control and †P Ͻ0.01vs.dexmedetomidine (50␮M)pretreatment by Scheffe’s F test.0.01,Fig.2).There was no significant difference be-tween the clonidine-and dexmedetomidine-induced decreases.In a time control study,the 7␮M rocuronium-induced twitch depression was not sig-nificantly altered by the sham drug (traces of modified Kreb’s solution).There was no significant difference among the 7␮M rocuronium-induced twitch depres-sions before the application of clonidine,dexmedeto-midine,or the sham drug (Fig.2)and that in the normal diaphragm in the experiment in which the rocuronium concentration–twitch tension relationship was investigated (Fig.1).DISCUSSIONThe results of the present study demonstrated that at supraphysiologic,but not therapeutic,concentrations,clonidine and dexmedetomidine enhanced rocuronium-induced neuromuscular block on indirectly elicited twitch tension of the rat diaphragm in vitro .We used a low stimulus frequency (0.1Hz)to avoid eliciting a frequency-dependent decrease in quantal acetylcholine release from the motor nerve terminal,which consequently induces fade of twitch tension.Therefore,the depression of indirectly elicited twitch tension caused by rocuronium is a reflection of com-petitive block at the postjunctional nicotinic acetylcho-line receptors.Rocuronium concentrations eliciting neuromuscular block in the normal diaphragm in the present study using adult rats (IC 50:8.038␮M)are about five times higher than those of a previous clinical study in which effects of rocuronium on adductor pollicis muscles in adult patients were inves-tigated (IC 50:1.565␮M;0.954␮g/mL)(9).Clonidine and dexmedetomidine were tested at a concentration near the therapeutic maximal free plasma concentra-tion (nanomolar range,0.05␮M;0.0133and 0.0118␮g/mL,respectively)(3,10–13)and at a concentration much higher than the therapeutic one (micromolarrange,50␮M;13.3and 11.8␮g/mL,respectively)to elicit these actions fully.Yohimbine was tested at an experimental concentration (50␮M)to fully elicit its ␣-2adrenoceptor blocking action.The rocuronium concentration–indirectly elicited twitch tension curve for the normal diaphragm was shifted leftward by 50␮M clonidine and 50␮M dexmedetomidine but was not shifted by 0.05␮M clonidine or 0.05␮M dexmedetomidine.Clonidine dexmedetomidine,and yohimbine (each 50␮M)had no effect on directly elicited twitch tensions,indicating that these ␣-2agonists/antagonist do not influence muscle contractility.These results indicate that high concentrations (micromolar order)of clonidine and dexmedetomidine enhance the neuromuscular block-ing action of rocuronium.Enhancing effects of clonidine and dexmedetomidine were also indicated by results showing that indirectly elicited twitch ten-sions partially depressed by 7␮M rocuronium were further depressed by clonidine and dexmedetomidine in concentration-dependent manner at concentrations of 30␮M or more.Clonidine-induced enhancement of nondepolarizing neuromuscular block was not de-tected in a previous study (8)under the condition of partial and progressing neuromuscular block,but we detected the enhancement under the condition of partial and stabilized neuromuscular block.The leftward shifts of the curves induced by 50␮M clonidine and 50␮M dexmedetomidine were not antagonized by coapplication of 50␮M yohimbine.Therapeutic maximal free plasma concentrations of clonidine and dexmedetomidine eliciting ␣-2agonist action are at most 0.003␮g/mL,i.e.,0.011␮M for dexmedetomidine and 0.013␮M for clonidine (3,11–13);however,0.05␮M clonidine and 0.05␮M dexmedetomidine did not influence the curve for the normal diaphragm.These results indicate that the enhancing effects of clonidine and dexmedetomidine on the rocuronium-induced neuromuscular block were not mediated by ␣-2receptors and support the results of previous studies showing that ␣-2adrener-gic stimulation,which presynaptically depresses exci-tatory synaptic transmission in the central nervous system (14–16),did not influence acetylcholine release from motor nerve terminals (5).Clonidine and dexme-detomidine also have very weak ␣-1activities;how-ever,the mechanisms mediating ␣-1receptors may not contribute to the enhancing effects of clonidine and dexmedetomidine on the rocuronium-induced neuromuscular block,because it has been shown that stimulation of ␣-1receptors on motor nerve terminals elicits an increase in acetylcholine release (17),which may rather antagonize,but not enhance,the neuro-muscular block.It has been reported that clonidine and yohimbine noncompetitively or competitively block neuronal-and muscle-type nicotinic acetylcholine receptors in vitro (6,7,18).It is likely that the clonidine-and yohimbine-induced leftward shifts of the curves were elicitedbyFigure 2.Effects of dexmedetomidine (n ϭ9),clonidine(n ϭ6),and sham drug (n ϭ6)on twitch tensions partially depressed by 7␮M rocuronium.*P Ͻ0.01versus 7␮M rocuronium alone by Scheffe ´’s F test.Data are expressed as mean Ϯsd .these cholinergic effects.Dexmedetomidine-induced block of nicotinic acetylcholine receptors has not been proved;however,it is possible because etomidate,which is one of the imidazoles related to dexmedetomidine and has an anesthetic action similar to that of dexme-detomidine,has been shown to noncompetitively and competitively block muscle-type nicotinic acetylcholine receptors(19).It is thought that the cholinergic effects of clonidine,dexmedetomidine,and yohimbine may also enhance actions of nondepolarizing neuromuscular blocking drugs other than rocuronium because postjunc-tional actions of nondepolarizing neuromuscular block-ing drugs are qualitatively similar to each other.The effects of clonidine and dexmedetomidine on nicotinic acetylcholine receptors,demonstrated as left-ward shifts of the rocuronium concentration–twitch tension curves and as enhancing effects on the7␮M rocuronium-induced partial neuromuscular block,did not appear at concentrations of nanomolar range, which is near their therapeutic free plasma concentra-tions(3,11–13),but were seen at concentrations of micromolar range,which is much greater than their therapeutic concentrations.It is suspected that clinical applications of clonidine and dexmedetomidine may not pharmacodynamically influence the clinical muscle re-laxation induced by nondepolarizing neuromuscular blocking drugs.␣-2agonist-induced changes in clinical muscle relaxations reported previously(1,3)may origi-nate from other pharmacokinetic mechanisms;e.g.,in-crease in plasma rocuronium concentration induced by dexmedetomidine(3).The results of the present study demonstrate that clonidine and dexmedetomidine at concentrations of 30␮M or more,which are not clinically relevant,but not at therapeutic nanomolar concentrations,pharma-codynamically enhance the rocuronium-induced neu-romuscular block mediating non-␣-2adrenergic mechanisms;i.e.,blocking actions on postjunctional nicotinic acetylcholine receptors.REFERENCES1.Nakahara T,Akazawa T,Kinoshita Y,Nozaki J.The effect ofclonidine on the duration of vecuronium-induced neuromuscu-lar blockade in humans.Masui1995;44:1458–63.2.Takahashi H,Nishikawa T.Oral clonidine does not alter vecu-ronium neuromuscular blockade in anaesthetized patients.Can J Anaesth1995;42:511–5.3.Talke PO,Caldwell JE,Richardson CA,Kirkegaard-Nielsen H,Stafford M.The effects of dexmedetomidine on neuromuscular blockade in human volunteers.Anesth Analg1999;88:633–9. 4.Weinger MB,Partridge BL,Henry AF.Dexmedetomidine doesnot modify the neuromuscular blocking action of vecuronium in the anaesthetized rat.Br J Anaesth1995;74:455–7.5.Snider RM,Gerald MC.Noradrenergic-mediated potentiationof acetylcholine release from the phrenic nerve:evidence for presynaptic alpha1-adrenoceptor involvement.Life Sci1982;31: 853–7.6.Chiou LC,Chang CC.Effect of clonidine on neuromusculartransmission and the nicotinic acetylcholine receptor.Proc Nat Sci Counc Repub China B1984;8:148–54.7.Musgrave IF,Krautwurst D,Schultz G.Imidazoline bindingsites and signal transduction pathways.Clin Exp Pharmacol Physiol1996;23:990–4.8.Banos J,Badia A,Jane F.Facilitatory action of adrenergic drugson muscle twitch evoked by nerve stimulation in the curarized rat phrenic hemidiaphragm.Arch Int Pharmacodyn Ther 1988;293:219–27.9.Saldien V,Vermeyen KM,Wuyts FL.Target-controlled infusionof rocuronium in infants,children,and adults:a comparison of the pharmacokinetic and pharmacodynamic relationship.Anesth Analg2003;97:44–9.10.MacDonald E,Scheinin M.Distribution and pharmacology ofalpha2-adrenoceptors in the central nervous system.J Physiol Pharmacol1995;46:241–58.11.Segal IS,Jarvis DJ,Duncan SR,White PF,Maze M.Clinicalefficacy of oral-transdermal clonidine combinations during the perioperative period.Anesthesiology1991;74:220–5.12.Bloor BC,Ward DS,Belleville JP,Maze M.Effects of intravenousdexmedetomidine in humans.II.Hemodynamic changes.Anes-thesiology1992;77:1134–42.13.Ebert TJ,Hall JE,Barney JA,et al.The effects of increasingplasma concentrations of dexmedetomidine in humans.Anes-thesiology2000;93:382–94.14.Martire M,Pistritto G,Mores N,et al.Presynaptic A2-adrenoceptors and neuropeptide Y Y2receptors inhibit[3H]no-radrenaline release from rat hypothalamic synaptosomes via different mechanisms.Neurosci Lett1995;188:9–12.15.Coelho JE,de Mendonca A,Ribeiro JA.Presynaptic inhibitoryreceptors mediate the depression of synaptic transmission upon hypoxia in rat hippocampal slices.Brain Res2000;869: 158–65.ner TA,Lee A,Aicher SA,Rosin DL.Hippocampal alpha2a-adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes.J Comp Neurol1998;395:310–27.17.Vizi ES.Evidence that catecholamines increase acetylcholinerelease from neuromuscular junction through stimulation of alpha-1adrenoceptors.Naunyn Schmiedebergs Arch Pharma-col1991;343:435–8.18.Abelson KS,Hoglund AU.The effects of the alpha2-adrenergic receptor agonists clonidine and rilmenidine,and antagonists yohimbine and efaroxan,on the spinal cholin-ergic receptor system in the rat.Basic Clin Pharmacol Toxicol 2004;94:153–60.19.Ziebell MR,Nirthanan S,Husain SS,et al.Identification ofbinding sites in the nicotinic acetylcholine receptor for[3H]azi-etomidate,a photoactivatable general anesthetic.J Biol Chem 2004;279:17640–9.。