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ANESTHETIC PHARMACOLOGY

Lack in Effects of Therapeutic Concentrations of Dexmedetomidine and Clonidine on the Neuromuscular Blocking Action of Rocuronium in Isolated Rat Diaphragms

Eichi Narimatsu, MD, PhD^*, Tomohisa Niiya, MD, PhD^*, Mikito Kawamata, MD, PhD^*, and Akiyoshi Namiki, MD, PhD^*

From the Departments of *Anesthesiology and Traumatology and Critical Care Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.

Address correspondence and reprint requests to Dr. Eichi Narimatsu, MD, PhD, Department of Anesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo 060-8543, Japan. Address e-mail to enarimat{at}sapmed.ac.jp.

	Abstract

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BACKGROUND: We investigated the effects of the -2 adrenoceptor 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 at 0.1 Hz were evaluated.

RESULTS: Clonidine and dexmedetomidine 50 µM (n = 6 each), but not 0.05 µM, shifted the rocuronium concentration–twitch tension curves to the left and decreased the rocuronium concentration for 50% twitch depression (IC₅₀) compared with control (n = 9, P < 0.01). The leftward shift induced by clonidine 50 µM or dexmedetomidine 50 µM was not antagonized by yohimbine 50 µM, an -2 adrenoceptor antagonist. Twitch tensions partially depressed by 7 µM rocuronium, to about 65% of the control, were further suppressed in a concentration-dependent manner by clonidine (n = 6) and dexmedetomidine (n = 9) at concentrations of 30 µM or more (P < 0.01).

CONCLUSIONS: These results indicate that very high, but not therapeutic, concentrations of clonidine and dexmedetomidine enhance the neuromuscular blocking action of rocuronium, but this is not mediated by an agonist action on -2 adrenoceptors.

	Introduction

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Concurrently administered drugs could affect the actions of nondepolarizing neuromuscular blocking drugs. Clonidine and dexmedetomidine, -2 adrenergic agonists, have been used in the perioperative period and in intensive care for sedation, analgesia, and management of hypertension. However, the effects of -2 adrenergic agonists on the actions of nondepolarizing neuromuscular blocking drugs have not been extensively studied.

The in vivo effects of -2 adrenergic 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 relaxation. Dexmedetomidine enhances rocuronium-induced muscle relaxation in volunteers (3) but does not significantly 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 release from motor nerve terminals (5). Clonidine depresses 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 hypothesis that -2 adrenergic 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.

	METHODS

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The study was approved by the Animal Care and Use Committee of Sapporo Medical University. Adult male Wistar rats (7–9 Wk old and weighing 235–270 g, n = 84) 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 diaphragm (10 mm 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 (25 mL in volume), fixed at the ribcage, inferiorly positioned and suspended at the central tendon from a force displacement transducer (FT-03, Grass, Quincy, MA) using a 3-0 silk suture. The chamber was filled with modified Kreb’s solution maintained at 27°C and bubbled with 95% O₂–5% CO₂. The composition of the modified Kreb’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 was 7.40 ± 0.05 while bubbling.

Twitch tension was elicited by indirect (phrenic nerve) or direct (muscle) supramaximal constant current stimulation at 0.1 Hz (for 0.05 and for 0.2 ms in the cases of indirect stimulation and direct stimulation, respectively) using a stimulator (S48, Grass) and a constant current unit with an optimal preload (2.5–5.0 g) to deliver maximal twitch tension. For indirect stimulation, the phrenic nerve was positioned on wire bipolar platinum electrodes and electrically stimulated. For direct stimulation, a diaphragm specimen, pretreated with a sufficient concentration (12 µM) of d-tubocurarine to completely abolish neuromuscular transmission, was positioned between a pair of plate bipolar platinum electrodes (each 25 mm x 12 mm) and electrically stimulated. The twitch tension was recorded via the force transducer on a thermal chart recorder.

After the twitch tension had been stabilized for at least 30 min, single twitch tension (averaged in groups of five) was measured and taken as a control. A study drug was then added to the bathing solution. After stabilization of the drug effect for at least 10 min, twitch tension was again measured. The drug concentrations were obtained by adding a freshly prepared solution (dissolved in modified Kreb’s solution) of rocuronium (100 µM or 10 mM), dexmedetomidine (5 or 400 µM), clonidine (5 µM or 5 mM), or yohimbine (5 mM) with calibrated micropipettes into 25 mL of modified Kreb’s solution in the tissue chamber. Relationships between rocuronium concentration and indirectly elicited twitch tension were investigated using control diaphragm strips with no pretreatment and those pretreated with dexmedetomidine (0.05 or 50 µM), clonidine (0.05 or 50 µM), yohimbine (50 µM), dexmedetomidine + yohimbine (50 µM each), or clonidine + yohimbine (50 µM each) (Table 1). To investigate the effects of dexmedetomidine and clonidine on rocuronium-induced twitch depression, dexmedetomidine (n = 9), clonidine (n = 6), or a sham drug (traces of modified Kreb’s solution, n = 6) was applied as supplement and in a stepwise manner after the 7 µM 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 applying 50 µM of one of these drugs (n = 6 each). 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 diaphragm preparation with modified Kreb’s solution (indirect stimulation) or that containing 12 µM 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 bromide was obtained from N.V. Organon (Oss, The Netherlands). Dexmedetomidine hydrochloride was obtained from Abbott Laboratories (Abbott Park, IL). Clonidine hydrochloride, yohimbine hydrochloride (an -2 adrenergic antagonist), d-tubocurarine (a nondepolarizing neuromuscular blocking drug), and all other drugs were purchased from Sigma (St. Louis, MO).

Twitch tension data (% of the control value) are expressed as means ± sd. Competition analysis data were determined from a four-parameter logistic sigmoidal 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 Prism 4 (GraphPad Software, San Diego, CA). Data of the 50% inhibitory concentrations of the curves (IC₅₀, in micromolar); i.e., the rocuronium concentration for 50% twitch depression, are expressed as means with 95% confidence intervals. Data of log IC₅₀ (in log µM) and slope at log IC₅₀ are expressed as means ± se. Statistical significance in IC₅₀s was calculated from log IC₅₀. One- or twoway repeated-measures analysis of variance (ANOVA) with post hoc (Scheffe’s F) testing and one-way factorial ANOVA with Scheffé’s F test were used for statistical comparison, and P < 0.05 was accepted as significant. A shift of a curve was regarded as significant when two-way repeated-measures ANOVA demonstrated a statistically significant difference between the corresponding curves.

	RESULTS

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There 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 (15.9–19.3 g) among the experimental groups. Neither dexmedetomidine, clonidine nor yohimbine (each 50 µM) significantly altered the indirectly elicited twitch tensions and directly elicited twitch tensions.

The effects of -2 agonists and an -2 antagonist on rocuronium-induced twitch depressions were investigated. Rocuronium reduced the indirectly elicited twitch tensions in the control and all pretreated diaphragms in a concentration-dependent manner (P < 0.01, Fig. 1). There was no significant difference between the curves or between the IC₅₀ values in the control diaphragm and that pretreated with 0.05 µM dexmedetomidine or 0.05 µM clonidine (Fig. 1 and Table 2). Pretreatment of the diaphragm with 50 µM clonidine, 50 µM clonidine + 50 µM yohimbine or 50 µM yohimbine significantly shifted the curve leftward from the curve for the control diaphragm (P < 0.01, Fig. 1A) and decreased IC₅₀s of those curves from that for the control diaphragm (P < 0.01, Table 2). Similar to the action of 50 µM clonidine, pretreatment of the diaphragm with 50 µM dexmedetomidine or with 50 µM dexmedetomidine + 50 µM yohimbine significantly shifted the curve leftward from the curve for the control diaphragm (P < 0.01, Fig. 1B) and decreased IC₅₀s of those curves from that for the control diaphragm (P < 0.01, Table 2). The curve and its IC₅₀ for the diaphragm pretreated with 50 µM dexmedetomidine + 50 µM yohimbine significantly shifted leftward from and was smaller than those for the diaphragm pretreated with 50 µM dexmedetomidine alone, respectively (P < 0.01, Fig. 1B and Table 2). There was no significant difference between the slopes at IC₅₀s of the curves for any of the control and pretreated diaphragms.

View larger version (20K): [in this window] [in a new window]   Figure 1. Concentration–twitch tension curves for rocuronium of isolated rat diaphragms pretreated with clonidine (A), dexmedetomidine (B) and/or yohimbine. Data are expressed as mean ± sd of nine and six experiments for control and pretreated diaphragms, respectively.

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 < 0.01, Fig. 2). The twitch tensions decreased by 7 µM rocuronium, to about 65% of the control, were further decreased in a concentration-dependent manner by dexmedetomidine or clonidine, and these significant decreases were observed at concentrations of 30 µM or more (P < 0.01, Fig. 2). There was no significant difference between the clonidine- and dexmedetomidine-induced decreases. In a time control study, the 7 µM rocuronium-induced twitch depression was not significantly altered by the sham drug (traces of modified Kreb’s solution). There was no significant difference among the 7 µM rocuronium-induced twitch depressions before the application of clonidine, dexmedetomidine, 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).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of dexmedetomidine (n = 9), clonidine (n = 6), and sham drug (n = 6) on twitch tensions partially depressed by 7 µM rocuronium. *P < 0.01 versus 7 µM rocuronium alone by Scheffé’s F test. Data are expressed as mean ± sd.

	DISCUSSION

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The 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 (0.1 Hz) 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 competitive block at the postjunctional nicotinic acetylcholine receptors. Rocuronium concentrations eliciting neuromuscular block in the normal diaphragm in the present study using adult rats (IC₅₀: 8.038 µM) 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 investigated (IC₅₀: 1.565 µM; 0.954 µg/mL) (9). Clonidine and dexmedetomidine were tested at a concentration near the therapeutic maximal free plasma concentration (nanomolar range, 0.05 µM; 0.0133 and 0.0118 µg/mL, respectively) (3,10–13) and at a concentration much higher than the therapeutic one (micromolar range, 50 µM; 13.3 and 11.8 µg/mL, respectively) to elicit these actions fully. Yohimbine was tested at an experimental concentration (50 µM) to fully elicit its -2 adrenoceptor blocking action.

The rocuronium concentration–indirectly elicited twitch tension curve for the normal diaphragm was shifted leftward by 50 µM clonidine and 50 µM dexmedetomidine but was not shifted by 0.05 µM clonidine or 0.05 µM dexmedetomidine. Clonidine dexmedetomidine, and yohimbine (each 50 µM) had no effect on directly elicited twitch tensions, indicating that these -2 agonists/antagonist do not influence muscle contractility. These results indicate that high concentrations (micromolar order) of clonidine and dexmedetomidine enhance the neuromuscular blocking action of rocuronium. Enhancing effects of clonidine and dexmedetomidine were also indicated by results showing that indirectly elicited twitch tensions partially depressed by 7 µM rocuronium were further depressed by clonidine and dexmedetomidine in concentration-dependent manner at concentrations of 30 µM or more. Clonidine-induced enhancement of nondepolarizing neuromuscular block was not detected 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 µM clonidine and 50 µM dexmedetomidine were not antagonized by coapplication of 50 µM yohimbine. Therapeutic maximal free plasma concentrations of clonidine and dexmedetomidine eliciting -2 agonist action are at most 0.003 µg/mL, i.e., 0.011 µM for dexmedetomidine and 0.013 µM for clonidine (3,11–13); however, 0.05 µM clonidine and 0.05 µM 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 -2 receptors and support the results of previous studies showing that -2 adrenergic stimulation, which presynaptically depresses excitatory synaptic transmission in the central nervous system (14–16), did not influence acetylcholine release from motor nerve terminals (5). Clonidine and dexmedetomidine also have very weak -1 activities; however, the mechanisms mediating -1 receptors 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 -1 receptors on motor nerve terminals elicits an increase in acetylcholine release (17), which may rather antagonize, but not enhance, the neuromuscular 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 elicited by 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 dexmedetomidine, 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 postjunctional actions of nondepolarizing neuromuscular blocking drugs are qualitatively similar to each other.

The effects of clonidine and dexmedetomidine on nicotinic acetylcholine receptors, demonstrated as leftward shifts of the rocuronium concentration–twitch tension curves and as enhancing effects on the 7 µM rocuronium-induced partial neuromuscular block, did not appear at concentrations of nanomolar range, which is near their therapeutic free plasma concentrations (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 relaxation induced by nondepolarizing neuromuscular blocking drugs. -2 agonist-induced changes in clinical muscle relaxations reported previously (1,3) may originate from other pharmacokinetic mechanisms; e.g., increase in plasma rocuronium concentration induced by dexmedetomidine (3).

The results of the present study demonstrate that clonidine and dexmedetomidine at concentrations of 30 µM or more, which are not clinically relevant, but not at therapeutic nanomolar concentrations, pharmacodynamically enhance the rocuronium-induced neuromuscular block mediating non--2 adrenergic mechanisms; i.e., blocking actions on postjunctional nicotinic acetylcholine receptors.

	Footnotes

 
Accepted for publication January 11, 2007.

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