Some Effects of d-Tubocurarine Alone and Combined with Halothane or Isoflurane on Neuromuscular Transmission

Abstract

The mechanisms contributing to the neuromuscular block produced by nondepolarizing muscle relaxants and inhaled anesthetics include:1) receptor blockade, 2) open or closed ion channel block, 3) decreased transmitter release, and 4) receptor desensitization. In this study we investigated the contributions of receptor and ion channel block. We used the two microelectrode voltage clamp to evaluate miniature end-plate currents (MEPCs) for amplitude and time constant of decay (tau) before and after the application of 1) d-Tubocurarine (DTC) (10^-7-10-6 M) alone, and then 2) the same concentration of DTC plus 0.5% or 1.0% halothane or isoflurane delivered by passing compressed air through a flow and temperature compensated vaporizer. The electrodes were maintained in the same cell for the entire experiment. DTC alone decreased MEPC amplitude to 91.3% +/- 4.5% and 65.1% +/- 5.6% of control at 10^-7 and 10^-6 M, respectively. MEPC amplitude with 10^-6 M DTC decreased further to 52.4% +/- 7.6% and 37.4% +/- 7.0% of control after the addition of 0.5% and 1.0% halothane, respectively. After the application of DTC 10^-7 M tau was 94.7% +/- 3.1% of control and decreased to 73.7% +/- 7.1% of control with 10^-6 M DTC. After the application of DTC 10 (^-6) M the addition of 0.5% and 1% halothane decreased tau to 52.4% +/- 7.6% and 30.0% +/- 5.9% of control. Isoflurane produced similar changes. This study provides evidence that at least some of the augmentation of nondepolarizing relaxants by inhaled anesthetics can be explained by the additive effect of nondepolarizing muscle relaxants and inhaled anesthetics on MEPC amplitude and tau.

(Anesth Analg 1995;81:763-7)

The ability of inhaled anesthetics to augment the effects of nondepolarizing muscle relaxants has long been recognized ^[1-5]. The mechanisms underlying this augmentation have been only partially explored. Thus, Karis et al. ^[6] noted that inhaled anesthetics decreased the amplitude of the miniature end-plate potential and Gage and Hamill ^[7], using either extracellular single microelectrode recording or intracellular two microelectrode voltage clamping techniques, noted the ability of these compounds to decrease the amplitude and time constant of decay (tau) of the miniature end-plate current (MEPC). This work was confirmed and extended by Sokoll et al. ^[8] who developed dose-response relationships between amplitude and tau of the MEPC for both halothane and isoflurane. In the studies by Gage and Hamill ^[7] and Sokoll et al. ^[8], the decrease in amplitude of the MEPC can be attributed to the decrease in tau.

The effects of nondepolarizing relaxants on neuromuscular transmission have long been attributed to the ability of these drugs to interfere with the actions of acetylcholine (ACh) in a competitive manner at the cholinergic receptor of the neuromuscular junction. This block of the ACh receptor produces a decrease in the MEPC amplitude. Gibb and Marshall ^[9], however, noted that, in addition to blockade of the postjunctional acetylcholine receptor, d-tubocurarine (DTC) also produced a modest block (approximately a 30% decrease of tau) of the acetylcholine receptor associated ion channel. In addition, both Gibb and Marshall ^[9] and Magleby et al. ^[10] documented an ability of DTC to augment rundown of trains of end-plate currents (EPCs) indirectly elicited at tetanic frequencies (50, 100, and 150 Hz).

The effects of potent inhaled anesthetics on the ion channel might add to the effects of DTC on both the receptor and receptor associated ion channel, thus producing a more intense block.

This study was performed to attempt to correlate the previously noted actions of DTC and potent inhaled anesthetics on the MEPC when administered alone and to attempt to correlate their combined actions at the neuromuscular junction. We administered DTC alone, and then combined with one of the two concentrations of the anesthetic being studied, in an attempt to elucidate possible postsynaptic mechanisms by which inhaled anesthetics enhance the actions of nondepolarizing muscle relaxants.

Methods

The protocol for these experiments was approved by the Animal Care and Use Committee of the University of Iowa. Frogs were placed in a refrigerator and, when a state of hypothermic anesthesia had been reached, were removed from the refrigerator, stunned, and pithed. The sartorius muscle was rapidly removed, stretched to approximately 125% of its resting length and pinned to the bottom of a bath having a volume of 1 mL. The bottom of the bath was lined with Sylgard Registered Trademark (Dow Corning, Midland, MI). The muscle was constantly bathed with rapidly flowing electrolyte (3 mL/min) having the following ionic composition: NaCl 116, KCl 1.9, CaCl₂ 1.0, NaH₂ PO₄ 1.0, Na₂ HPO₄ 1.0 mM ^[10,11]. Fresh bathing solution entered one end of the bath and was removed by suction from the other.

Control recordings of MEPCs were made. The concentration of DTC to be studied was then applied to the muscle for 15 min and a second group of MEPCs was recorded. Previous experiments revealed that this duration of application permitted DTC to have its full effect on the surface cells being studied. After this, the inflowing electrolyte was switched to a separate reservoir containing the same concentration of DTC in electrolyte solution that was bubbled and equilibrated with the concentration of inhaled anesthetic selected for study. The inhaled anesthetic was applied by passing compressed air through a flow- and temperature-compensated vaporizer. After this mixture had been applied for 15 min MEPCs were again recorded. Each study was performed on one end-plate and both microelectrodes were kept in the cell throughout the study. The solution flowing into the bath was controlled by stopcocks close to the tissue bath. At the end of each study a sample of the electrolyte in the bath was obtained and analyzed for halothane or isoflurane using the method described by Davis ^[11].

A complete description of the technique of two microelectrode voltage clamping is published elsewhere ^[8]. Muscles from 200 frogs were studied. Satisfactory data were obtained from 10 cells for each concentration of DTC used. Data were used from one cell of each muscle. Briefly, at least 15 MEPCs were recorded from each cell for each of the recording periods. MEPCs were analyzed for duration of the growth phase, amplitude, and tau using a specially written computer program. The values determined for each of the 15 MEPCs of each experiment were converted to a single mean value for construction of the linear regression relationships. Ten cells were studied at each dose of DTC. Thus values of 150 individual MEPCs were used to determine each point of the regression. Values determined after the application of DTC and the DTC-halothane (or DTC-isoflurane) mixture were expressed as percentage of the predrug value. The concentrations of DTC studied were 10^-7, 3 times 10^-7, 5 times 10^-7, and 10^-6 M. Halothane and isoflurane were studied as delivered with the vaporizer set at 0.5% and 1.0%. The observations obtained were analyzed by linear regression and the construction of 95% confidence limits. For numerical calculations, DTC concentrations of 10^-7 and 10^-6 M were represented as 1 and 10, respectively.

Results

The concentrations of halothane in the bath fluid as determined by gas-liquid chromatography at vaporizer settings of 0.5% and 1.0% were 24.4 +/- 2.7 and 51.4 +/- 8.2 micro gram/mL whereas those for isoflurane were 33.7 +/- 3.9 and 72.4 +/- 6.8 micro gram/mL, respectively.

The effects of DTC and DTC plus halothane are seen in Figure 1. DTC alone at a concentration of 10^-7 M decreased the amplitude of the MEPC to 91.3% +/- 4.5% of control whereas DTC 10^-6 M resulted in a further decrease to 65.1% +/- 5.6% of control (P < 0.01) (y = -2.97x + 93.84). The addition of halothane at a vaporizer setting of 0.5% caused a further decrease in amplitude to 76.6 +/- 5.9 and 52.4 +/- 7.6, respectively (P < 0.01) at DTC concentrations of 10^-7 and 10^-6 M (y = -3.22x + 85.22). When the halothane vaporizer is set at 1.0% the amplitude of the MEPC is 37.4% +/- 7.0% of the control value after the application of the halothane-DTC 10^-6 M combination (y = -3.10x + 95.01 for DTC and y = -3.72x + 73.42 for DTC + 1% halothane).

Figure 1: Upper left, Effect of d-tubocurarine DTC alone and combined with halothane (hal) 0.5% on miniature end-plate current (MEPC) amplitude. Lower left, Effect of DTC alone and combined with 0.5% halothane on tau (TAU). Upper right, The effect of DTC alone and combined with halothane 1.0% on MEPC amplitude. Lower right, Effect of DTC alone and combined with 1% halothane on tau. The lines are derived by least-squares linear regression with 95% confidence limits denoted by the area included in the hatched lines.

The ability of DTC to block the ACh receptor associated ion channel is seen in Figure 1. At DTC concentrations of 10^-7 and 10^-6 M the value of tau was 94.7% +/- 3.1% and 73.1% +/- 7.1% of the control value, respectively (y = -2.35x + 98.09). This observation is similar to that of Gibb and Marshall ^[9] in the cut rat diaphragm. The addition of halothane 0.5% and 1.0% reduced tau further to 52.4% +/- 2.7% and 30.0% +/- 5.9% of control when combined with DTC 10^-6 M (y = -2.68x + 79.14). The ability of halothane and other potent inhaled anesthetics to produce a decrease in tau has been previously reported ^[7,8].

The effects of isoflurane are similar to those seen with halothane. The addition of DTC 10^-7 M and 10^-6 M resulted in a decrease in MEPC amplitude to 93.8% +/- 3.5% and 64.2% +/- 6.9% of control, respectively (y = -3.30x + 97.41). With the isoflurane vaporizer set at 1.0% the MEPC amplitude was further reduced to 70.0% +/- 6.4% and 34.6% +/- 7.6% of the control values (y = -4.013x + 74.70). Figure 2. As with halothane, isoflurane produced a dose-related decrease in tau which augmented that produced by DTC alone Figure 2.

Figure 2: Upper left, Effects of d-tubocurarine (DTC) alone and combined with isoflurane (ISF) 0.5% on miniature end-plate current (MEPC) amplitude. Lower left, Effect of DTC alone and combined with isoflurane 0.5% on tau (TAU). Upper right, The effect of DTC alone and combined with 1.0% isoflurane on MEPC amplitude. Lower right, The effects of DTC alone and combined with 1% isoflurane on tau. The lines are derived by least-squares linear regression with 95% confidence limits denoted by the area included in the hatched lines.

Discussion

The ability of ether to augment the effect of DTC was reported by Cullen ^[1,2]. In our study we have investigated the relative contribution of DTC and halothane or isoflurane to the total decrease in MEPC amplitude and time constant of decay. Our aim was to determine whether the combined effects of these drugs represented addition or potentiation.

Gage et al. ^[7] demonstrated the ability of inhaled anesthetics to decrease both the amplitude and tau of the MEPC. The decrease in amplitude can be explained as being related to the decrease in tau. By definition, tau is the time constant of decay of the MEPC and provides an estimate of the mean channel open time of that MEPC. The actual open times of the postsynaptic ACh receptor associated ion channels vary widely. The open time of these channels can vary from more than 1 ms to as short as 50 mu s. Channels with this short open time are commonly seen in patch clamp experiments ^[12,13]. Some channels may have even shorter open states, but these are not usually measured because the sampling time most commonly used in patch clamp experiments is 50 mu s. Thus, channels having an open state shorter than 50 mu s would be missed. The duration of the growth phase of the MEPC is usually less than 0.8 ms. Those channels having open times of less than the duration of the growth phase will contribute little, if anything, to the amplitude of the MEPC. The addition of a drug which decreases the mean channel open time will increase the number of channels whose open time is less than that of the growth phase and should result in a decrease in the amplitude of the current. If a significant number of channels of a single junction shift from having an open phase greater than the duration of the growth phase to one where they are shorter than the duration of the growth phase the result will be a decrease in the MEPC amplitude.

The ability of DTC to decrease miniature end-plate potential and MEPC amplitude by occupation of the cholinergic receptor is well accepted. DTC is also reported to decrease the open time of the acetylcholine activated ion channel ^[9]. Inhaled anesthetics might add to the effect of DTC by three mechanisms. The first is to decrease the open time of the ion channel. Halothane produces this effect ^[8,12]. The second is to decrease in some manner, the sensitivity of the cholinergic receptor to the neurotransmitter. This action, which would produce a decrease in MEPC amplitude with no change in channel open time, has not been reported nor was it noted in this study.

DTC decreases MEPC amplitude. We previously reported that halothane or isoflurane alone also decreases MEPC amplitude ^[8]. This decreased amplitude is more when DTC and halothane or isoflurane are applied simultaneously. Does this represent addition or potentiation?

In our previous study we observed that a 1% concentration of halothane (58.2 micro gram/mL) produced approximately a 28% decrease in MEPC amplitude and that the ED₅₀ for halothane-induced decrease in MEPC amplitude was 118 micro gram/mL ^[8]. This decrease in MEPC amplitude is similar to that seen with the addition of halothane to the DTC-depressed preparation. Thus, the effects of DTC and halothane appear to be additive. The same relationship applies to isoflurane.

In a separate set of experiments we examined the effect of halothane on the amplitude and quantum content of EPCs and the mobilization rate of quanta in the indirectly stimulated rat diaphragm ^[15]. Halothane decreased all of these values. Halothane alone increased rundown of a tetanic train of EPCs elicited at 40 Hz probably as a result of its decrease of rate of mobilization. It did not, however, produce run down of potentials induced ionophoretically at 40 Hz. This indicates that halothane does not produce desensitization of the nicotinic cholinergic receptor. These prejunctional effects of halothane can augment the relaxant effects of DTC.

If the previous analysis of the mechanism by which inhaled anesthetics augment the effects of muscle relaxants is correct, then barbiturate anesthetics should also cause some degree of increased action. Torda and Gage ^[14] reported that thiobarbiturates and several of other intravenously administered anesthetics also decrease the value of tau in a dose-related manner similar to that of inhaled anesthetics. Thus, if the present concept of augmentation is correct, then thiobarbiturates should also cause some increase in the effect of nondepolarizing relaxants. In clinical studies, dose-response relationships in patients are usually determined after the administration of anesthesia. Thus, in clinical studies one of two sequences is followed: In the first, the patient is anesthetized with a thiobarbiturate or other intravenously administered anesthetic after which increasing doses of muscle relaxant are administered to obtain a dose-response relationship. In the second sequence, the patient is anesthetized with thiobarbiturate followed by the administration of a potent inhaled anesthetic. Increasing doses of muscle relaxant are then administered to establish a second dose-response relationship. Under these circumstances, the administration of an inhaled anesthetic is associated with a more profound neuromuscular block compared to that seen where the intravenous anesthetic is administered alone. The muscle relaxant is always administered after the intravenous anesthetic. As a result, it is usually stated that the intravenously administered drug does not potentiate the effect of relaxants whereas potentiation is seen with the inhaled anesthetic. This may not be correct. Evaluation of the combined effects of muscle relaxants and anesthetics in humans or animals presents some problems. It would be preferable to establish the effect of the relaxant in the absence and then presence of the anesthetic. However, producing paralysis without anesthesia in experiments on either humans or animals presents significant ethical problems. The usual technique is to administer muscle relaxants with either barbiturate or inhaled drugs and compare the effects of the muscle relaxant with the different anesthetics. This technique produces only comparative information. Additional information might be obtained by anesthetizing the patient or animal with an inhaled anesthetic and establishing a level of neuromuscular block, after which a range of doses of thiopental or other intravenous drugs could be administered and the effect on the neuromuscular block observed. This would produce information concerning the interactions of the muscle relaxant and intravenously administered drug on a background of inhaled drug effects. This type of experiment has not been done in humans or even in in vitro preparations. It is possible that intravenously administered drugs may potentiate the effects of relaxants, but to a different degree than inhalationally administered anesthetics. This type of study should be performed with an in vitro system.

A second possible explanation lies in the fact that all dose-response relationships determined entail the use of indirect stimulation of the neuromuscular junction. It is possible that the inhaled anesthetics might decrease release of transmitter from the nerve terminal whereas the intravenous drugs have little or no effect. This problem is currently under investigation in our laboratory.

This study has investigated the interactions between DTC and halothane and isoflurane at the neuromuscular junction of the frog to attempt to elucidate the mechanisms underlying the increased action of non-depolarizing relaxants by inhaled anesthetics. We have demonstrated that both halothane and isoflurane augment the effects of DTC by producing a decrease in MEPC amplitude. This decreased MEPC amplitude is the result of a decrease in tau which results from administration of the anesthetic.

The data provided in this study will not alter the clinical practice of muscle relaxant use. It does help to explain one of the mechanisms underlying the increased action of nondepolarizing relaxants when used in conjunction with inhaled anesthetics.