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Anesth Analg 2000;90:970-974
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

Pentobarbital Has Curare-Like Effects on Adult-Type Nicotinic Acetylcholine Receptor Channel Currents

Krampfl Klaus, MD, Schlesinger Friedrich, BSc, Dengler Reinhardt, MD, and Johannes Bufler, MD

Neurological Department of the Medical School Hannover, Hannover, Germany

Address correspondence and reprint requests to Johannes Bufler, MD, Neurological Department of the Medical School Hannover, 30623 Hannover, Germany. Address e-mail to Bufler.Johannes @MH-Hannover.de.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Pentobarbital (PB) is widely used as a short-term sedative and anticonvulsive drug with a side-effect of relaxing muscle tone. We investigated block of nicotinic acetylcholine receptor (nAChR) channel currents by PB using the patch-clamp technique in combination with an ultrafast system for solution exchange. As a preparation, recombinant rat adult-type nAChR channels transiently expressed in HEK293 cells were used. Appli-cation of 1 mM acetylcholine to small cells or outside-out patches showed a transient current with fast activation and desensitization kinetics. Adding PB to the acetylcholine-containing solution resulted in a decrease of the time constant of current decay and of the peak current amplitude starting at concentrations >0.01 mM PB. Preincubation of nAChR channels with PB led to a decrease of the peak current amplitude without alteration of activation and desensitization kinetics caused by competitive block of nAChR channels. In conclusion, similar to the effect of d-Tubocurarine, block of nAChR channel currents by PB can be explained by a combination of open-channel and competitive block.

Implications: The interaction between adult-type nicotinic acetylcholine receptors, acetylcholine, and pentobarbital was biophysically investigated by using the patch-clamp technique in combination with tools for ultrafast solution exchange. PB elicited open-channel block and competitive block of nicotinic acetylcholine receptor channel currents, whereas the latter seems to be effective in clinically relevant concentrations.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Barbiturates have several therapeutic effects, including induction of sleep, suppression of epileptic seizures, and reduction of muscle tone (1). They have multiple effects on different receptors of the central and peripheral nervous system. Several types of ion channels have been described as possible targets for these compounds, including {gamma}-aminobutyric acid type A-receptor channels (2), voltage dependent Ca++- channels (3,4) and nicotinic acetylcholine receptor channels (5).

During the last few years, we investigated block of native rat embryonic-type nicotinic acetylcholine receptor (nAChR) channels by different compounds, such as local anesthetics (6), cholinesterase inhibitors (6), noncompetitive muscle relaxants (7), benzodiazepines (8), volatile anesthetics (9,10), and ketamine (11). These studies demonstrated that block of nAChR channels by chemically different compounds has two different mechanisms: open-channel block and competitive block of the receptor. In open-channel block, blockers bind to channels already opened by the agonist. Competitive block means that the blocker interferes with acetylcholine (ACh) binding sites in the resting state of the receptor. Experimentally, these two different mechanisms of block were differentiated by using tools for ultrafast application of agonists. With the experimental data, complex kinetic schemes of activation, desensitization, resensitization, and block of embryonic-type ({gamma}) nAChR channels (6,7,12) were constructed, and binding rate constants and unbinding and isomerization rates were determined quantitatively.

We investigated the molecular interaction between mouse muscle adult-type {epsilon}-nAChR channels and ACh and pentobarbital (PB). In contrast to previous studies performed on native {gamma}-nAChR channels, the present experiments were done at recombinant ({epsilon}) nAChR channels expressed in HEK293 cells. Dilger et al. (13) investigated the block of {gamma}-nAChR channels expressed from BC3H-1 cells by PB and barbital and found a combination of both open-channel and competitive block of nAChR channels, whereas PB showed higher affinity to nAChR channels. We wanted to test if interactions between {epsilon}-nAChR channels, ACh, and PB occurred in clinically relevant concentrations. Pharmacologically, we focused on two questions: 1) Is it possible to distinguish different block mechanisms of nAChR channels by PB, and 2) Does block of {epsilon}-nAChR channels follow similar principles like block of {gamma}-nAChR channels?


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Transformed human embryonic kidney 293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO2/95% air incubator. For transfection, cells were suspended in a buffer containing: 50 mM K2HPO4 and 20 mM K-acetate, pH 7.35. For co-transfection of mouse {alpha}-, ß-, {delta}-, and {epsilon}-nAChR subunits, the corresponding cDNA was added to the suspension. To visualize transfected cells, they were co-transfected with cDNA of green fluorescent protein. For transfection, an electroporation device by EquiBio (Kent, UK) was used. Transfected cells were replated on glass coverslips and incubated for 15–24 h before recording.

Transfected cells were visualized with an upright microscope (Axioscop; Zeiss, Oberkochen, Germany) at x400 magnification. Experiments were performed at room temperature (20°C), and cells were continuously superfused with extracellular solution containing: 162 mM NaCl, 5.3 mM KCl, 0.6 mM Na2HPO4, 0.22 mM KH2PO4, 15 mM HEPES, and 5.6 mM glucose. pH was adjusted to 7.4 with NaOH. Patch pipettes were pulled from thin-walled borosilicate glass tubing with filament (Clark, Pangbourne, UK) by using a DMZ-Universal puller (Zeitz, Augsburg, Germany). Pipette tips were filled with high K + solution (140 mM KCl, 2 mM MgCl2, 11 mM EGTA, 10 mM glucose; pH 7.4) and had a resistance between 8 and 12 M{Omega}. Outside-out patches were obtained by using standard methods (14). Currents were recorded with an Axopatch 200B amplifier, digidata 1200 interface, and pCLAMP 6.4 software (Axon Instruments, Foster City, CA). Data were stored to hard disk with a sampling frequency of 20 kHz and filtered for analysis at 5 kHz. Five to 10 single-current traces were averaged for analysis. Desensitization was fitted with Chebyshev and Simplex methods. Experimental data were given as mean ± SEM. Unless otherwise stated, all drugs were obtained from Sigma (St. Louis, MO). ACh and PB (Synopharm, Barsbuettel, Germany) were dissolved in extracellular solution before each experiment.

Fast application of agonist was done by using a piezo-driven device for concentration-clamp measurements (15) adapted to an upright microscope setup (16). A smooth liquid filament was achieved with a single outflow (glass tubing 0.15-mm inner diameter). Between pulses, patches were bathed in a continuously flowing background solution. Time for solution exchange was <100 µs measured with an open pipette and high electrolyte gradient (6,16).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
If antagonists have binding sites at the open state of a receptor, this type of block is denoted as an open-channel block. By using fast application techniques, open-channel block of nAChR channels by PB was demonstrated convincingly by applying pulses of agonist and antagonist together. As shown in Figure 1 (upper trace) application of a saturating concentration of 1 mM ACh to an outside-out patch containing {epsilon}-nAChR channels resulted in a transient current similar to that measured after application of ACh to {gamma}-nAChR channels (see Refs. 6,7). The current rose within 0.6 ms (time interval between 10% and 90% of the total response was measured) to the peak current amplitude of -78 pA (corresponding to the simultaneous opening of at least 30 single channels) and declined thereafter monoexponentially in the presence of ACh with a time constant of current decay, {tau}, of 41 ms in this experiment as a result of desensitization of {epsilon}-nAChR channels. The addition of PB to the ACh-containing test solution had two different effects: 1) decrease of the peak current amplitude and 2) decrease of {tau} with increasing concentrations of PB. In the experiment of Figure 1, the peak current amplitude decreased from -78 pA with no PB to -38 pA when a large concentration of 1 mM PB was added. {tau} decreased approximately 10-fold from 41 ms with no PB (upper trace) to 4.3 ms with 1 mM PB added to the test solution (lower trace on Fig. 1, see inset, application of 1 mM ACh + 1 mM PB shown at an expanded time scale). The current decay in presence of ACh + PB was monoexponential at all concentrations, indicating one binding site of PB at the receptor down to very small current amplitudes (the straight line shows the quality of the monoexponential fit; see inset of Fig. 1).



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  		Figure 1. Averaged currents from one outside-out patch of HEK293 cells after transfection of cDNA of adult-type nicotinic acetylcholine receptor ({epsilon}-nAChR) channels activated by 1 mM acetylcholine (ACh) and pentobarbital (PB) with concentrations as indicated. Every single-current trace is the average of 5–10 single traces. The inset below the lower current trace shows the onset of the current with higher time resolution. The monoexponential fit of current decay caused by open-channel block is added to that current trace. Holding potential, -40 mV.

 
Figure 2 shows data of seven independent open-channel block experiments. {tau} in the presence of PB and ACh decreased from 47.5 ± 8.2 ms (n = 7) to 6.2 ± 2.4 ms (n = 7) between 0.01 and 1 mM PB, respectively and had a value of 2.0 ± 1.2 ms (n = 7, Fig. 2A) at 10 mM PB. The relative peak current amplitude decreased little up to a concentration of 0.3 mM PB and reached a relative peak current amplitude of 0.43 ± 0.11 (n = 5) at 10 mM PB (Fig. 2B).



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  		Figure 2. Dose-response curves for the current decay time constant {tau} (A) and the relative peak current amplitude (B) with increasing concentrations of pentobarbital (PB). The data points represent the average of seven independent experiments ± SEM.

 
If outside-out patches were preincubated with PB before the application of ACh, the antagonist could equilibrate with the unliganded state of the receptor. To test for PB binding at the unliganded nAChR, outside-out patches were preincubated with different PB concentrations via the background flow of the fast application system, and 200-ms pulses of 1 mM ACh were applied with a frequency of 0.1 Hz. Original data of such an experiment were depicted in Figure 3A. One hundred twenty-five repetitive 1-mM ACh pulses were applied to an outside-out patch before, during, and after incubation with different concentrations of PB as indicated. There was a concentration-dependent decrease of the peak current amplitude. It decreased to around 10% of control when 3 mM PB was applied. The blockade was nearly completely reversible after washout of PB. For example, in the experiment shown in Figure 3A, the peak current amplitude after washout of 3 mM PB summarized to -49 pA, compared with -53 pA in the beginning of the experiment, resulting in a run down of 7.5%. Experiments with run down >10% were excluded from analysis. Activation and desensitization kinetics were not altered after preincubation with PB (not shown) as already demonstrated for d-Tubocurarine (7). Therefore, receptors with bound PB were not available for activation by ACh, but receptors with no PB bound were functionally not altered.



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  		Figure 3. A, Amplitudes of single pulses of 1 mM acetylcholine (ACh) to one outside-out patch from HEK293 cells containing adult-type nicotinic acetylcholine receptor ({epsilon}-nAChR) channels before, during, and after incubation with different concentrations of pentobarbital (PB) are plotted versus number of ACh pulses. 200-ms pulses were applied with a frequency of 0.1 Hz. PB was applied (concentration as indicated) via the background flow of the fast application system. B, Dose-response curves at different concentrations of PB added to the background solution of three independent experiments.

 
Dose-inhibition curves after incubation of {epsilon}-nAChR channels with increasing PB-concentrations were shown in the diagram of Fig. 3B (three independent experiments of the type shown in Fig. 3A). Preincubation of outside-out patches with 3 mM PB resulted in a block of the peak current amplitude to 8% of control. From the diagram (Fig. 3B), the dissociation constant, for PB at nAChR channels could be read. It was between 15 and 30 µM.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Concentration-clamp techniques are very powerful tools for investigating kinetic processes underlying activation of ligand-gated channels and the analysis of the molecular interactions between drugs, ligands, and receptors. We have investigated different drugs interfering with activation of nicotinic channels, and the mechanisms of block of nAChR channels by different compounds could be analyzed by two basic mechanisms: open-channel block and competitive block (6–11).

Barbiturates are known to interact with different states of the nicotinic receptor. Liu and Madsen (17) showed that pentobarbitone reduced the open time of the {gamma}-nAChR channels of chicken myotubes. Similar results were published by Jacobson et al. (18) on nicotinic receptor channels expressed in bovine chromaffin cells. Dilger et al. (13) supposed a model wherein PB binds to both open and closed states of the nAChR channels, however, preferentially to the open state of the receptor.

In a previous study, it was shown that d-Tubocurarine binds to both the open and closed state of nAChR channels (7). Binding and unbinding rates necessary to describe the interaction between d-Tubocurarine, ACh, and nAChR channels were experimentally determined and integrated in a circular reaction scheme. The probability of different states in the scheme being occupied with time could be calculated (7). In contrast to open-channel block by physostigmine or procaine (6), open-channel block by d-Tubocurarine was characterized by a monoexponential time course of current decay down to very small current amplitudes. The unbinding rate constant of the blocker from the open state of the receptor determined the ratio of the current amplitude decaying as a result of block on the whole current amplitude. The lower the value of the unbinding rate, the lower the remaining current amplitude decaying via the desensitization route (see Ref. 6, Fig. 2A). Therefore, open-channel block of nAChR channels is mainly characterized by different unbinding rates from the open state of the receptor, e.g., the unbinding rate of physostigmine from the open state of {gamma}-nAChR channels was 200/s [as found for open-channel block of {gamma}-nAChR channels by PB (13)], whereas d-Tubocurarine unbound much slower with 0.8/s (7). PB had the same characteristics of open-channel block at {epsilon}-nAChR channels like d-Tubocurarine at {gamma}-nAChR channels, i.e., monoexponential decay of the ACh-activated current down to low steady state values (Fig. 1), increasing time constants of current decay with decreasing concentrations of PB (Figs. 1, 2A) and relatively little reduction of the peak current amplitude also at high concentrations of PB (Figs. 1, 2B).

By using our fast application system, the test-solution containing ACh or ACh plus PB and the background solution containing Ringer’s solution with or without PB could be changed independently during an experiment. We were therefore able to simulate the situation of postsynaptic {epsilon}-nAChR channels under the influence of PB in vivo. As was expected for competitive block, the time necessary for PB binding to the unliganded state of the receptor decreased with increasing concentrations of PB, and the extent of block increased (Fig. 3A). As was suggested for the action of d-Tubocurarine at nAChR channels (7), competitive block by PB seems to be more important in causing reduction of muscle tone than open-channel block (see dose-response diagram in Figs. 2B and 3B). However, comparing dose inhibition curves for d-Tubocurarine and PB with the dissociation constant of 160 nM (7) and 15–30 µM, respectively, showed that block of nAChR channels by PB was at least two orders of magnitude less effective than competitive block by d-Tubocurarine. In contrast to the data shown by Dilger et al. (13), we observed a pronounced reduction of the peak current amplitude after preincubation of patches with PB (Fig. 3). One reason for these experimental differences may be different affinities of PB to open or closed states of {epsilon}- or {gamma}-nAChR channels.

During the IV administration of PB, serum concentrations of 200 µM PB were measured (19). PB ~56% are protein bound (20). Therefore, free drug concentration can be estimated to ~88 µM under therapeutic conditions. At this concentration range, open-channel block of {epsilon}-nAChR channel currents by PB was not effective (Fig. 2). However, peak current amplitude was reduced to >50% of control after preincubation of nicotinic receptors with PB concentrations > 30 µM reflecting competitive block (Fig. 3). Because of the safety factor of neuromuscular transmission, normal postsynaptic signals might be generated even if 75% of postsynaptic receptors were not available for activation (21). Therefore, competitive block of {epsilon}-nAChR channels by PB may add to central nervous mechanisms leading to the reduction of muscle tone during treatment with PB.

Our study is the first to investigate the effect of PB on molecular well defined {epsilon}-nAChR channels. We conclude from our data that similar block mechanisms, as postulated for {gamma}-nAChR channels, hold true for {epsilon}-nAChR channels. Two different mechanism of block of {epsilon}-nAChR channels by PB were distinguished: open-channel block and competitive block; the latter seems to be clinically relevant in causing muscle relaxation.


   Acknowledgments
 
Supported by grants of the Deutsche Forschungsgemeinschaft (Bu938/2–1,2–2,3–1).

The authors wish to thank S. Henke for expert technical assistance, U. Jensen for help with transfection-experiments and H. Lochmüller for providing us with nAChR-cDNA.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication December 29, 1999.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press