Anesthesiology, Article

Pralidoxime inhibits paraoxon-induced depression of rocuronium-neuromuscular block in a time-dependent fashion

Unlabelled imageAmerican Journal of Emergency Medicine (2012) 30, 901-907

Original Contribution

Pralidoxime inhibits paraoxon-induced depression of rocuronium-neuromuscular block in a time-dependent fashion?,??

Eichi Narimatsu MD, PhD a,b,?, Tomohisa Niiya MD, PhD b, Kazunobu Takahashi MD b,

Masanori Yamauchi MD, PhD b, Michiaki Yamakage MD, PhD b

aDepartment of Traumatology and Critical Care Medicine, Sapporo Medical University School of Medicine,

Sapporo 060-8543, Japan

bDepartment of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan

Received 22 April 2011; revised 16 June 2011; accepted 23 June 2011

Abstract

Objective: The composite effects of organophosphorus (OP)-cholinesterase (ChE) inhibitors and oximes on the actions of nondepolarizing Neuromuscular blockers in acute OP-ChE inhibitor intoxication have not been evaluated in detail. We investigated the effects of paraoxon (Pox) (an OP-ChE inhibitor) and pralidoxime (PAM) (an oxime) on the nondepolarizing neuromuscular blocking action of rocuronium. Methods: Isometric twitch tensions of rat left phrenic nerve-hemidiaphragm preparations elicited by indirect (phrenic nerve) supramaximal stimulation at 0.1 Hz were evaluated. Analysis of variance with post hoc testing was used for statistical comparison, and P b .05 was accepted as significant.

Results: Rocuronium reduced the indirectly elicited twitch tensions in normal (50% inhibitory concentration [IC50], 9.84 [9.64-10.04] uM, mean [95% confidence interval]) and all pretreated diaphragms (P b .01, n = 6) in a concentration-dependent fashion. Paraoxon caused a rightward shift in the rocuronium concentration-twitch tension curve (IC50, 15.48 [15.24-15.72] uM). The rightward shift was completely inhibited by previous copretreatment (IC50, 9.98 [9.77-10.20] uM) and partially inhibited by simultaneous copretreatment (IC50, 11.68 [11.45-11.91] uM) with PAM but was not inhibited by subsequent copretreatment (IC50, 13.69 [13.39-13.99] uM) with PAM (P b .01, n = 6). Atropine did not influence the rightward shift (P b .01, n = 6).

Discussion: Paraoxon depressed rocuronium-induced neuromuscular block by inhibiting ChEs, and the action of Pox was inhibited by PAM. Pralidoxime acts more intensely when applied earlier. The time- dependent effect of PAM indicates that the preceding presence of PAM in proximity to ChEs before Pox is necessary for definite suppression of the Pox-induced ChE inhibition.

(C) 2012

? Source(s) of support in the form of equipment, drugs, or grants: This work was supported by a Grant-in-Aid for Scientific Research (C) (19592093) from the Japan Society for the Promotion of Science and by a departmental fund of Sapporo Medical University School of Medicine.

?? The name of organization and date of assembly if the article has been presented: In the 2008 Annual Meeting, American Society of Anesthesiologists,

October 18 to 22, 2008 Orlando (Narimatsu E, Niiya T, Kawamata T, Namiki A, and Asai Y; Effects of an organophosphate and an oxime on the neuromuscular blocking action of rocuronium).

* Corresponding author. Departments of Anesthesiology and Critical Care Medicine, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo

060-8543, Japan. Tel.: +81 11 611 2111×3568; fax: +81 11 631 9683.

E-mail address: [email protected] (E. Narimatsu).

0735-6757/$ – see front matter (C) 2012 doi:10.1016/j.ajem.2011.06.013

Introduction

acute intoxication with organophosphorus (OP)-cholin- esterase (ChE) inhibitors, which are used in agricultural (insecticides and acaricides), hygienic, and military settings (nerve agents), causes various life-threatening symptoms in the central, peripheral, and autonomic nervous systems [1-4]. Organophosphorus-ChE inhibitors covalently bind to an esteric site, one of the acetylcholine (ACh)-binding sites on ChEs; this binding is irreversible [2,3]. The accumulation of extracellular ACh in the synaptic cleft induced by ChE inhibition causes dysfunction of these nervous systems by overstimulating nicotinic and muscarinic ACh receptors [5]. Organophosphate-induced ChE inhibition causes dysfunction at the neuromuscular junction, inducing spasms, fascicula- tions, weakness, and paralysis of skeletal muscles [2,4,6].

Nondepolarizing neuromuscular blockers are often used as Muscle relaxants for emergency care, intensive care, and anesthesia for patients with acute OP-ChE inhibitor intoxica- tion, for example, for the facilitation of tracheal intubation and mechanical ventilation or for the symptomatolytic treatment of Muscle spasm, fasciculation, or convulsion. cholinesterase inhibition attenuates the actions of nondepolarizing neuro- muscular blockers by inducing the accumulation of ACh because the blockers are competitive nicotinic ACh receptor antagonists [7]. Neostigmine and edrophonium, reversible ChE inhibitors, have been widely used for the clinical reversal of a nondepolarizing neuromuscular block [8].

Oximes have been used therapeutically for acute OP-ChE inhibitor intoxication [1,2]. Oximes reactivate ChE function by removing bound OP molecules [3]. The efficacy of this process increases, associated with earlier oxime application

[9] and longer aging half-life of OP compounds [3]. It is presumed that oximes inhibit the OP-ChE inhibitor-induced attenuation of the actions of nondepolarizing neuromuscular blockers and that oximes administrated earlier elicit more intense effectiveness; however, these presumptions have not yet been proven.

This study was designed to directly test the hypothesis that OP-ChE inhibitors attenuate the actions of nondepolar- izing neuromuscular blockers and that oximes inhibit this attenuation. We investigated the in vitro effects of paraoxon (Pox) (an OP-ChE inhibitor, active metabolite of parathion) and the composite effects of Pox and PAM (an oxime) on the neuromuscular ability of rocuronium to inhibit indirectly elicited twitch tension.

Methods

Animal preparation

The study was approved by the Animal Care and Use Committee of Sapporo Medical University. Male Wistar rats (5-6 weeks old and weighing 155-185 g, bred to a 12:12

light-dark schedule [lights on at 0600 hour]) with water and food provided ad libitum, n = 54) were euthanized by excessive sevoflurane anesthesia in oxygen and then exsanguinated. Left and right hemidiaphragms with attached phrenic nerve were removed rapidly, keeping the central tendon and rib cage intact. Strips of the left and right diaphragms with the phrenic nerve attached, which had been cut parallel to the muscle fibers (10 mm in width), were then dissected. Each isolated strip was mounted vertically in a glass tissue chamber (25 mL in volume), inferiorly fixed at the rib cage, 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 Krebs solution maintained at 33?C and bubbled with 95% O2 to 5% CO2. The composition of the modified Krebs 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;

pH was 7.40 +- 0.05 during bubbling.

Twitch tension measurement

Twitch tension was elicited by indirect (phrenic nerve) or direct (muscle) supramaximal constant current stimulation at

0.1 Hz (for 0.05 and 0.2 milliseconds in the cases of indirect stimulation and direct stimulation, respectively) using a stimulator (S48; Grass) and a constant-current unit with an optimal preload to deliver maximal twitch tension. For indirect stimulation, the left phrenic nerve connected to the left diaphragm specimen was positioned on wire bipolar platinum electrodes and was electrically stimulated. For direct stimulation, the right diaphragm specimen was positioned between a pair of plate bipolar platinum electrodes (each 25 x 12 mm) and electrically stimulated. To completely abolish neuromuscular transmission in the right diaphragm specimens, a sufficient concentration (5 uM) of d-tubocura- rine was added to modified Krebs solution when direct stimulation was performed. The twitch tension was recorded via the force transducer on a thermal chart recorder.

Drug application

After twitch tension had been stabilized for at least 20 minutes, baseline single-twitch tension (averaged in groups of 6) was measured and taken as a control for the preparation. Then, a study drug was added to the bathing solution. After stabilization of the drug effect for at least 5 minutes, twitch tension (averaged in groups of 6) was measured again and taken as a drug effect. Drug application and measure- ment were repeated when necessary. The drug concentra- tions were obtained by adding freshly prepared solutions, rocuronium dissolved to 1 mmol/L in modified Krebs solu- tion and other drugs for pretreatment, which were dissolved at a concentration of 1000x the final concentration in modi- fied Krebs solution (except Pox) or in dimethyl sulfoxide (DMSO; for Pox). Treatment solutions were administered

using calibrated micropipettes into 25 mL of modified Krebs solution in the tissue chamber (volume of modified Krebs solution or DMSO necessary to achieve a pretreatment level of 0.025 mL and final concentration of DMSO in modified Krebs solution of 0.1%). Rocuronium bromide was obtained from N.V. Organon (Merck & Co, Inc, Whitehouse Station, NJ). Paraoxon (0.02 uM, O,O-diethyl O-p-nitrophenyl phosphate, an OP-ChE inhibitor) was obtained from Chem Service (West Chester, PA). Pralidoxime (10 uM, 2- pyridinealdoxime methochloride, an oxime) was obtained from Acros Organics (Geel, Belgium). Atropine (Atr; 10 uM, a nonselective muscarinic ACh receptor antagonist), DMSO (vehicle for Pox), and all other drugs were purchased from Sigma (St Louis, MO).

Before investigating the action of rocuronium, the effects of pretreatments, which involved 1, 2, or 3 steps of treatment and/or washout (WO), on indirectly and directly elicited twitch tensions were evaluated (Fig. 1). The pretreatments consisted of (1) modified Krebs solution without drugs (Normal group), (2) Pox alone and then WO (Pox/WO group), (3) Pox followed by Pox + PAM and then WO (Pox/ Pox + PAM/WO group), (4) Pox + PAM and then WO (Pox

+ PAM/WO group), (5) PAM followed by Pox + PAM and then WO (PAM/Pox + PAM/WO group), (6) PAM alone (PAM group), (7) Atr alone (Atr group), (8) Pox followed by WO and then atropine (Pox/WO/Atr group), and (9) DMSO alone (DMSO group). The Pox/Pox + PAM/WO, Pox + PAM/WO, and PAM/Pox + PAM/WO groups denote subsequent, simultaneous, and preceding additions of PAM to Pox, respectively. The period required for each pretreat- ment step was 5 minutes or more. When Pox was applied, subsequent WO was performed by full exchanges with modified Krebs solution (containing 5 uM d-tubocurarine only in the case of direct stimulation) 5 times to evaluate Pox-induced irreversible ChE inhibition. WO was not performed when drugs with reversible actions were used for pretreatment. The rocuronium-induced changes in indirectly elicited twitch tension were subsequently investi- gated by supplementing rocuronium in a stepwise fashion; the related control values were defined as the baseline single-twitch tensions in the same preparation recorded before estimating drug effects.

Data analysis

Competition analysis data were obtained from a 4-parameter logistic sigmoidal dose-response model (all values being used for analysis) fitted to the rocuronium concentration-indirectly elicited twitch tension curves using the computer program Prism 5 (GraphPad Software, Inc, San Diego, CA). Twitch tension data (percentage of the control value) are expressed as means +- SDs. Data of the 50% inhibitory concentrations of the curves (IC50; in micromolar), that is, the rocuronium concentration for 50% twitch depression, are expressed as means with 95% confidence

Measurement with no study drug (control twitch tension)

Pretreatment

Groups

Steps of pretreatments

n

Step I

Step 2

Step 3

Normal

mKrebs

6

Pox/ WO

Pox

WO

6

Pox/ Pox+PAM/ WO

Pox

Pox+PAM

WO

6

Pox+PAM/ WO

Pox+PAM

WO

6

PAM/ Pox+PAM/ WO

PAM

Pox+PAM

WO

6

PAM

PAM

6

Atr

Atr

6

Pox/ WO/ Atr

Pox

WO

Atr

6

DMSO

DMSO

6

Measurement of drug effect for pretreatment (indirect and direct stimulation)

Estimations of rocuronium concentration-twitch tension relationships (indirect stimulation)

Fig. 1 Summary of the study design for drug application. Effects of pretreatments, involving 1, 2, or 3 steps of drug application or WO on indirectly and directly elicited twitch tensions, were evaluated. Each pretreatment step was 5 minutes or longer. After each pretreatment, rocuronium concentration-indirectly elicited twitch tension relationship was investigated. Pox was 0.02 uM, an OP-ChE inhibitor; PAM, 10 uM, an oxime; Atr, 10 uM, a nonselective muscarinic ACh receptor antagonist; DMSO, 0.1%. WO was performed after the application of Pox. The volume of vehicle for pretreatments (modified Krebs solution for drugs except Pox; DMSO for Pox) was 0.025 mL. mKrebs sol indicates modified Krebs solution.

intervals. Data of log IC50 (in log micromolar) and slope at log IC50 are expressed as means +- SEs. Statistical significance in IC50 was calculated based on log IC50. Different specimens were used in each experiment. Data were accepted only when twitch tension returned to 95% to 105% of the value observed immediately before the application of rocuronium (for indirect stimulation) or before the application of the drug solutions used for pretreatment (for direct stimulation) by rinsing the diaphragm specimen with modified Krebs solution (for indirect stimulation) or that containing d- tubocurarine (5 uM, for direct stimulation). The twitch tension was blindly measured by researchers who had not been informed of the experimental group to which each specimen belonged.

One- or 2-way repeated-measures analysis of variance (ANOVA) and 1-way factorial ANOVA with post hoc (Bonferroni/Dunn) testing were used for statistical compar- ison, and P b .05 was accepted as significant. A shift in the curve was regarded as significant when 2-way repeated- measures ANOVA demonstrated a statistically significant difference between the corresponding curves.

Results

Six rats were used in each experimental group. No diaphragm specimen was eliminated from the study, and there was no exclusion of data from the results in this study. Among the experimental groups, there were no significant differences in body weights of rats, in sizes of diaphragm specimens (width and length), or in indirectly or directly elicited twitch tension before the pretreatments (6.9-13.1 g). The preloads necessary to deliver maximal twitch tension for left (for indirect stimulation) and right (for direct stimulation) diaphragm specimens were 3.7 +- 0.8 g (2.3-4.9 g, n = 54) and

3.8 +- 0.9 g (2.1-5.0 g, n = 54), respectively. After treatment with d-tubocurarine (5 uM), only direct (muscle) stimulation but not indirect (nerve) stimulation elicited twitch tension.

Effects of Pox, PAM, and Atr on diaphragms

Treatments of the diaphragm with modified Krebs solution without drugs (normal group), simultaneous Pox + PAM (Pox + PAM/WO group), PAM then Pox + PAM (PAM/Pox + PAM/WO group), PAM alone (PAM group), Atr alone (Atr group), or DMSO alone (DMSO group) (Fig. 1)

did not significantly alter the indirectly elicited twitch tensions; subsequent application of rocuronium (0.1 uM) also failed to alter twitch tensions (Fig. 2A-C, n = 6). Treatments of the diaphragm with Pox alone (Pox/WO group), Pox then Pox + PAM (Pox/Pox + PAM/WO group), and Pox then Atr (Pox/ WO/Atr group) (Fig. 1) increased indirectly elicited twitch tensions to approximately 130% to 140% of the control level (P b .01), and the increased twitch tensions were not significantly decreased after WO (Fig. 2B-D, n = 6). Subsequent application of rocuronium (0.1 uM) decreased the magnitude of these increased twitch tensions to the control level (Fig. 2B-D, P b .01, n = 6). None of these treatments significantly altered the directly elicited twitch tensions (n = 6).

Effects of Pox, PAM, and Atr on the action of rocuronium on the diaphragm

Rocuronium reduced the indirectly elicited twitch ten- sions in the normal group and all other groups in a concentration-dependent manner (Fig. 3, P b .01, n = 6). The IC50 and slope at IC50 of the rocuronium concentration- twitch tension curve in the normal group were 9.84 uM (9.64-10.04 uM) and -5.194 +- 0.233, respectively (Table 1).

A B

175

150

125

100

75

50

25

0

1Pox*

1WO*

2Pox+PAM* 2WO

Normal PAM DMSO

Atr

1Pox/ WO

2Pox+PAM/ WO

Cont Step 1

Step 1

+ Roc (0.1 ?M)

Cont Step 1 Step 2

D

Step 2

+ Roc (0.1 ?M)

3Pox*

1Pox+PAM*

3Atr*

1WO*

10 g

1Pox*

3

WO*

2PAM

2Pox+PAM 2WO

60 sec.

1Pox/ Pox+PAM/ WO

2PAM/ Pox+PAM/ WO

3Pox/ Wo/ Atr

Twitch tension (% of the control)

Twitch tension (% of the control)

175

150

125

100

75

50

25

0

C

Twitch tension (% of the control)

175

150

125

100

75

50

25

0

Cont Step 1 Step 2 Step 3

Step 3

+ Roc (0.1 ?M)

Cont

Pox 0.02 ?M WO Roc 0.1 ?M

Fig. 2 Effects of pretreatments on indirectly elicited twitch tension in isolated rat diaphragms with Pox (0.02 uM, an OP-ChE inhibitor), PAM (10 uM, an oxime), Atr (10 uM, a nonselective muscarinic ACh receptor antagonist), and/or DMSO (0.1%, vehicle for Pox). The details of the pretreatments are shown in Fig. 1. The control values were defined as baseline single-twitch tensions in the same preparation, which were recorded before drug application. Graph A shows the effects of pretreatments involving 1 step with “modified Krebs solution (normal),” “PAM,” “DMSO,” or “Atr.” Graph B shows the effects of pretreatments involving 2 steps with “Pox and then WO” or “Pox + PAM and then WO.” Graph C shows the effects of pretreatments involving 3 steps, which were achieved with “Pox, Pox + PAM and then WO”; “PAM, Pox + PAM and then WO”; or “Pox, WO and then Atr.” A representative change in the twitch tension induced by Pox and then WO (Pox/WO group) is shown in graph D. Data are expressed as means +- SD of 6 experiments and percentage of the control value (before drug application). Asterisk indicates P b .01 vs control. Cont indicates control values.

Fig. 3 Rocuronium concentration-indirectly elicited twitch tension curve in isolated rat diaphragms with no drug pretreatment (normal diaphragm) and those pretreated with Pox (0.02 uM, an OP-ChE inhibitor), PAM (10 uM, an oxime), Atr (10 uM, a muscarinic ACh receptor antagonist), and/or DMSO (0.1%, the vehicle for Pox). Details of the pretreatment conditions are shown in Fig. 1. The control values were defined as baseline single-twitch tensions in the same preparation and were recorded before pretreatments. Data are expressed as the means +- SD of 6 experiments and percentage of the control value (before pretreatment).

Pretreatment with Pox alone (Pox/WO group) and Pox then Pox + PAM (Pox/Pox + PAM/WO group) shifted the curve rightward and increased the IC50 as compared with

Table 1 The IC50 and slopes at the IC50 points of rocuronium concentration-indirectly elicited twitch tension curves for normal diaphragm and diaphragms pretreated with Pox (0.02 uM), PAM (10 uM), Atr (10 uM), and/or DMSO (0.1%)

those in the normal group (Fig. 3A and B and Table 1, P b .01, n = 6). There were no significant differences in the curve or IC50 between the Pox/WO group and Pox/Pox +

Groups for pretreatments

Log IC50 (log uM)

IC50 (uM)

Slope at IC50

Normal

0.992 +- 0.004

9.84 (9.64-10.04)

-5.194 +- 0.233

Pox/WO

1.190 +- 0.004 ?

15.48 (15.24-15.72)

-6.568 +- 0.300 +

Pox/Pox + PAM/WO

1.136 +- 0.005 ?,?

13.69 (13.39-13.99)

-6.522 +- 0.385 +

Pox + PAM/WO

1.067 +- 0.004 ?,?,||

11.68 (11.45-11.91)

-5.226 +- 0.243 ?

PAM/Pox + PAM/WO

0.999 +- 0.005 ?,||,#

9.98 (9.77-10.20)

-5.171 +- 0.253 ?,?

PAM

0.997 +- 0.004 ?,||,#

9.94 (9.76-10.12)

-5.193 +- 0.212 ?,?

Atr

0.991 +- 0.005 ?,||,#

9.79 (9.58-10.02)

-5.176 +- 0.261 ?,?

Pox/WO/Atr

1.186 +- 0.003 ?,||,#,++

15.36 (15.14-15.58)

-6.554 +- 0.271 +,??,??

DMSO

0.990 +- 0.005 ?,||,#

9.77 (9.57-9.98)

-5.162 +- 0.235 ?,?

Values are expressed as means with the 95% confidence interval for IC50 (micromolar), means +- SE for log IC50 (log micromolar), and slope at log IC50 for 6

experiments. The statistical significance of IC50 was calculated from log IC50.

* P b .01 vs the normal group.

+ P b .05 vs the normal group.

? P b .01 vs the Pox/WO group.

? P b .05 vs the Pox/WO group.

|| P b .01 vs the Pox/Pox + PAM/WO group.

? P b .05 vs the Pox/Pox + PAM/WO group.

# P b .01 vs the Pox + PAM/WO group.

?? P b .05 vs the Pox + PAM/WO group.

++ P b .01vs the Atr group.

?? P b .05 vs the Atr group.

PAM/WO group (Fig. 3A and B and Table 1). Pretreatment with simultaneous Pox + PAM (Pox + PAM/WO group) shifted the curve rightward and increased the IC50 in comparison with the normal group, but the shift and increase were significantly less than those observed for the Pox/Pox + PAM/WO group and Pox/WO group (Fig. 3A and B and Table 1, P b .01, n = 6). Pretreatment with PAM then Pox + PAM (PAM/Pox + PAM/WO group), PAM alone (PAM group), Atr alone (Atr group), or DMSO alone (DMSO group) did not shift the curve and did not alter the IC50 in comparison with the normal group (Fig. 3 and Table 1, n = 6). Pretreatment with Pox then Atr (Pox/WO/Atr group) shifted the curve rightward and increased the IC50 in comparison with those observed for the Atr and normal groups (Fig. 3A and D and Table 1, P b .01, n = 6). Pretreatment with Pox alone (Pox/WO group), Pox then Pox + PAM (Pox/Pox + PAM/WO group), and Pox then Atr (Pox/WO/Atr group) increased the slope of the curve at the point that represented the IC50 (Table 1, P b .05, n = 6).

Discussion

The results of the present study demonstrated that Pox shifted the rocuronium concentration-indirectly elicited twitch tension curve rightward and that the rightward shift was completely inhibited by previous copretreatment and partially inhibited by simultaneous copretreatment with PAM but was not inhibited by subsequent copretreatment with PAM.

Paraoxon (0.02 uM) alone, which inhibits about 95% of the activity of true-type ChE; E.C.3117 [10], facilitated indirectly elicited twitch tension probably through repetitive firings on muscle fibers induced by ChE inhibition-induced repetitive end plate potentials in response to single nerve stimuli, in agreement with previous results [11]. The Pox- induced twitch facilitation was inhibited by rocuronium (0.1 uM) but not by Atr, indicating that the nicotinic but not muscarinic mechanism controls the twitch facilitation. The depression of Pox-induced twitch facilitation induced by rocuronium (0.1 uM), which had no effect on twitch tension in the normal group, was advantageous for strictly analyzing the rocuronium concentration-indirectly elicited twitch tension curves; this process revealed a plateau phase that preceded the steepest portion of the curve. The effect of Pox, which is irreversible, is maintained in ChEs in the diaphragm specimens after WO; therefore, the results from the specimens reflect residual ChE activity. None of the pretreatments altered directly elicited twitch tension, which reflects muscle contractility, indicating that the pretreatment- induced changes in the action of rocuronium originated from changes in neuromuscular transmission.

Rocuronium dose dependently depressed indirectly elicited twitch tension in the normal diaphragm, reflecting competitive block by rocuronium on the postjunctional nicotinic ACh receptors but not its prejunctional actions because a low

stimulus frequency (0.1 Hz) does not elicit prejunctional effects of rocuronium, that is, a frequency-dependent depres- sion of quantal ACh release from the motor nerve terminal and a consequent twitch fade [12-14]. In a single skeletal muscle fiber, rocuronium-induced twitch depression reflects a failure in the approach of end plate potentials to the threshold-eliciting action potentials in an all-or-none fashion. However, in a diaphragm strip, a bundle of many muscle fibers, it reflects a decrease in the proportion of twitching fibers.

Paraoxon shifted the rocuronium concentration-indirectly elicited twitch tension curves rightward; the rightward shift was not influenced by Atr. Atropine alone and DMSO alone did not influence the curve. These results indicate that extracellular ACh accumulated by Pox-induced ChE inhibition attenuates the rocuronium-induced twitch depression by antagonizing the competitive nicotinic ACh receptor block [7], which is essentially the same mechanism that underlies the usual reversal of nondepolarizing neuromuscular block achieved using neostigmine or edrophonium. These results also show that muscarinic ACh receptor-mediated mechanisms, M1 receptor-mediated facilitation, and M2 receptor-mediated depression of ACh release from the motor nerve terminals

[15] do not influence the attenuation. About 95% of the ChE inhibition induced by Pox (0.02 uM) [10] reduced the rocuronium-induced profound and intermediate neuromu- scular block (about 7% and 52% of control at 15 and 10 uM rocuronium, respectively; normal group) to only approximately 57% and barely 93% of control, respectively (Pox/WO group), supporting the fact that neostigmine cannot fully reverse profound nondepolarizing neuromuscular block [16]. The Pox- induced rightward shift accompanied an increase in the slope at IC50, reflecting facilitation of the rocuronium-induced block at postjunctional nicotinic ACh receptors. It is possible as the mechanism that Pox binds to ACh receptors as do other OPs [17], inducing receptor desensitization, which may enhance the action of rocuronium.

Inhibition of the Pox-induced rightward shift by previous copretreatment with PAM (PAM/Pox + PAM/WO group) indicates that PAM, which reaches the extracellular space around ChEs before Pox, can depress Pox-induced ChE inhibition. These findings support the results of a previous study showing that oximes protect ChEs from OPs by slowing down the rate of phosphorylation [3,18]. Subsequent copretreatment with PAM (Pox/Pox + PAM/WO group) did not influence the Pox-induced rightward shift (Pox/WO group), indicating that PAM, which has reached ChEs after Pox, cannot depress the Pox-induced ChE inhibition. The ineffectiveness of delayed PAM treatment strongly suggests that, in this experimental condition, before PAM has reached ChEs, Pox (0.02 uM)-induced ChE inhibition progresses to the aging reaction; spontaneous dealkylation of OP mole- cules bind to ChEs, and PAM becomes less effective [3]. Pralidoxime alone did not shift the curve, indicating that its weak and reversible ChE inhibitory action [19] does not significantly contribute to the rightward shift. Simultaneous copretreatment with Pox and PAM (Pox + PAM/WO group)

had an intermediate effect; the Pox-induced rightward shift was significant but was reduced in magnitude compared with that induced by Pox with subsequent copretreatment with PAM (Pox/Pox + PAM/WO group). These results indicate that PAM, which has reached ChEs together with Pox, only partially inhibits the Pox-induced ChE inhibition. These time-dependent effects of PAM indicate that the presence of PAM around ChEs before Pox administration is necessary for definite suppression of Pox-induced ChE inhibition. The results of a previous study showed similar time-dependent effects: the application of PAM before, but not after, application of Pox inhibited the Pox-induced depression of CA1-field excitatory postsynaptic potentials in rat hippo- campal slices [20].

This in vitro study was designed to simulate the clinical neuromuscular action of rocuronium under the condition of parathion intoxication, which produces Pox in vivo as an active metabolite, with PAM medication. It is presumed clinically that OP-ChE inhibitors attenuate nondepolarizing Muscle relaxation and that oximes inhibit attenuation; however, to the best of our knowledge, few clinical quantitative data for these actions have been reported in published articles. Accordingly, determination of the appropriate doses of nondepolarizing neuromuscular blockers for patients with acute OP-ChE inhibitor intoxication has been lacking. The time-dependent decrease in the efficacy of PAM suggests that nondepolarizing neuromuscular blockers should be applied at more than their usual doses for patients with OP-ChE inhibitor intoxication because the patients are usually treated later with oximes than with OP exposure. The time-dependent effect of PAM also supports results of previous studies demonstrating the reduced effectiveness of delayed oxime application [9], little benefit of PAM [21,22] for clinical OP intoxication, and the protective effects of pretreated oximes in the context of experimental OP nerve agent intoxication [23]. The Pox-induced twitch facilitation, probably reflecting clinical OP-induced spasms, is depressed by an infraeffective concen- tration of rocuronium (0.1 uM), which supports the results of a previous clinical study showing that low-dose pancuronium inhibited the OP-induced repetitive discharge of muscle fibers [24], which elicited spasms [11].

In conclusion, the results of the present study indicate that Pox-induced depression of the rocuronium-induced neuro- muscular block is inhibited by PAM. The efficacy of this reaction depends on the time of application, with increasing efficacy associated with earlier applications.

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