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Anesth Analg 1999;88:1406
© 1999 International Anesthesia Research Society

GENERAL ARTICLES

Depressant and Convulsant Barbiturates Both Inhibit Neuronal Nicotinic Acetylcholine Receptors

Itaru Watanabe, MD, Tomio Andoh, MD, PhD, Ryosuke Furuya, MD, Toshio Sasaki, MD, Yoshinori Kamiya, MD, and Hideki Itoh, MD

Department of Anesthesiology, Yokohama City University School of Medicine, Yokohama, Japan

Address correspondence and reprint requests to Tomio Andoh, MD, PhD, Department of Anesthesiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Address e-mail to tandoh{at}med.yokohama-cu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates.

Implications: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are widely expressed in the central and peripheral nervous systems, including autonomic ganglia (1,2). Both ganglionic and central neuronal nAchRs are very sensitive to a number of different anesthetics (3–6). Barbiturates strongly inhibit the neuronal nAchR-mediated current in adrenal medullary cells (3), the rat pheochromocytoma cell line (PC12 cells) (7), and molluscan neurons (8). Therefore, neuronal nAchRs are potential sites responsible for barbiturate anesthesia.

Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity (9). Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic actions, S(+)MPPB exhibits pure excitatory effects, including convulsion. S(+)MPPB exerts no sedative effect at any dose (10). 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid (CHEB) is another example of a convulsant barbiturate (11). We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current in PC12 cells to explore the possibility that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. If the inhibitory effects on neuronal nAchRs correlate to in vivo depressive effects on the central nervous system (CNS), it would be likely that modulation of neuronal nAchRs by barbiturates is relevant to their anesthetic action.

We conducted the experiment using PC12 cells, which express ganglion-type neuronal nAchRs, resembling those in postganglionic sympathetic neurons (12), because we have already studied the effects of an anesthetic barbiturate, thiopental, in this cell line (7). We assumed that the receptors in PC12 cells and the CNS have similar sensitivities to barbiturates because thiopental reportedly inhibits both central-type and ganglion-type receptors at clinically relevant concentrations (7,13).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
PC12 cells were cultured as previously described (7). Cells were not treated with nerve growth factors to avoid the influence of {gamma}-aminobutyric acid type A (GABAA) receptor activation by barbiturates. Cells were plated on collagen and poly-L-lysine–coated cover slips and used after an additional 2- to 4-day culture.

Membrane currents were recorded by using the whole cell voltage clamp method (14) under the conditions described previously after a slight modification (7). Cells on the cover slips were placed in a recording bath with an approximate volume of 1.5 mL and were continuously perfused at the rate of 1–2 mL/min with a standard external solution containing (in mM), NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, HEPES 10, glucose 11.1 (pH adjusted to 7.4 with NaOH). Heat-polished patch pipettes had a tip resistance of 3–7 M{Omega} when filled with an intracellular solution containing (in mM) CsCl 150, HEPES 10, EGTA 5, ATP-Mg 2 (pH 7.3 with CsOH). Cells were voltage-clamped at -60 mV with a patch clamp amplifier (CEZ 2400; Nihon Koden, Tokyo, Japan). Whole cell currents were filtered at 0.2 kHz with a Bessel filter and digitized at 1 kHz. Data were stored and analyzed on a microcomputer using pCLAMP software and Axograph (Axon Instruments, Foster City, CA). All experiments were performed at room temperature (22–25°C).

Nicotine 30 µM in the external solution was applied to cells using a rapid application technique described as the "Y-tube" method (15). This method enabled the complete exchange of the external solution surrounding the cell around 100 ms, as estimated by recording the liquid junction current produced at an open patch pipette. Nicotine, with or without the barbiturates, was applied for 4 or 5 s, and each application was separated by 5 min. For preincubation with the barbiturates, the external solution containing the drugs was perfused at the rate of 5 mL/min for 5 min before rapid application. Cells were perfused with the plain external solution at the same rate for 5 min to washout the drugs from the bath after the measurement.

The stereoisomers of MPPB were kindly provided by Prof. J Knabe (Saarland University, Saarbrüken, Germany). S(+) and R(-)MPPB were dissolved in 0.1 N NaOH to make a 30-mM solution just before the experiments. They were diluted with the external solution to the designated concentration. CHEB was from Tocris cookson (Bristol, UK), which was prepared in the same way as MPPB isomers.

We measured the peak and the nondesensitized current, which was defined as the average of the preceding 50 points at 4 s during agonist application. Because nicotine-induced currents declined with each application of nicotine, the response in the presence of the barbiturates was compared with the average of the elicited currents before and after the addition of the barbiturates. This procedure was rationalized by the findings that the second response was almost the same as the average of the first and third responses when nicotine was applied successively three times at a interval of 5 min (7). To avoid bias from cell to cell differences in the sensitivity to the barbiturates, we compared the effects of MPPB isomers in the same cells that were exposed to the isomers in a random sequence.

The decaying phases of the nicotine-induced current were fitted to either a single or a double exponential function of the following form by using the simplex method using Axograph software: Go


where I is the total peak current, Ifinal is the residual current at the steady-state condition, Ii is the peak current amplitude of the each component, and {tau}i is the time constant of the corresponding component. Goodness of fit was compared by using a {chi}2 test between single and double exponential models. This analysis was performed for the experiments with preincubation and for those in which nicotine alone was applied successively. The time constant ratio was defined as the ratio of {tau}i in the presence of the drugs relative to the average pre- and postcontrol. Desensitization was also evaluated by calculating the percent decay of the current (% current decay) defined by the following equation (16): Go


Data are expressed as mean ± SEM Statistical analysis was performed by using paired a t-test for comparisons between MPPB isomers. Analysis of variance, followed by Dunnett's test, was used for comparisons among different doses of the barbiturates. P <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Nicotine 30 µM elicited inward currents that decayed rapidly during nicotine application due to desensitization at the membrane potential of -60 mV in PC12 cells. Neither MPPB isomers nor CHEB alone produced any current responses in the tested cells at 100 µM. Both the depressant and convulsant isomer of MPPB inhibited the nicotine-induced inward current reversibly at 30 µM when co-applied with nicotine (Fig. 1A). Both R(-) and S(+)MPPB accelerated the current decay, resulting in the greater effects on the nondesensitized current than on the peak current. The acceleration of current decay by MPPB isomers was also observed when the cells were pretreated with the barbiturates for 5 min before the co-application of nicotine and MPPB isomers, which suggests that the acceleration of current decay is not due to slow onset of inhibition. The inhibition by MPPB isomers was dose-dependent, and the isomers at 100 µM almost completely blocked the current responses (Fig. 1B). As for potency of the MPPB isomers, the convulsant isomer showed slightly but significantly stronger inhibition than the depressant isomer at 30 µM. However, there was no significant difference between the concentrations for 50% inhibition (IC50 values) for these two isomers. The IC50 values of the peak current inhibition were 41.0 ± 1.4 µM for R(-) isomer and 35.0 ± 3.2 µM for S(+) isomer; those of the steady current inhibition were 19.2 ± 4.9 and 16.3 ± 2.4 µM for R(-) and S(+) isomers, respectively (Fig. 2).



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  		Figure 1. Inhibition of the nicotine induced current by 30 µM (A) and 100 µM (B) 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) isomers. The records are the evoked currents in a single PC12 cell voltage-clamped at -60 mV. The cell was exposed to nicotine 30 µM and nicotine plus R(-) or S(+)MPPB sequentially. The right panel of the upper row and the left panel of the lower row depict the same response. Drug application is indicated by the horizontal bar. S(+)- and R(-)MPPB were co-applied with nicotine without preincubation. At 30 µM, both MPPB isomers suppressed the nicotine-induced current and accelerated the current decay reversibly. The suppressive effect was greater on the nondesensitized current than on the peak current (A). Both R(-) and S(+) isomers of MPPB almost completely abolished the nondesensitized current at 100 µM in a different single cell (B).

 


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  		Figure 2. Concentration-inhibition curves for 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) isomers on the peak (A) and nondesensitized current (B). The currents in the presence of MPPB isomers were normalized to the average of the control currents before and after the addition of MPPB and were plotted against the concentration of the isomers. A least-squares fit was performed using the equation: I = 1 - Cn/(Cn + IC50n), where I = relative current, C = concentration of MPPB, n = the Hill coefficient, and IC50 = the concentration for 50% inhibition. Both isomers inhibited the nicotine-induced current in a dose-dependent manner. The fitting procedure gave IC50 values of 41 ± 1.4 and 35.0 ± 3.2 µM and Hill coefficients of 2.0 ± 0.12 and 2.09 ± 0.39 for the peak current inhibition by R(-) and S(+) isomers, respectively (A). It gave IC50 values of 19.2 ± 4.9 and 16.3 ± 2.4 µM and Hill coefficients of 1.04 ± 0.28 and 1.39 ± 0.26 for the nondesensitized current inhibition by R(-) and S(+) isomers, respectively (B). Inhibition by S(+) isomer, which is the convulsant isomer, was slightly but significantly stronger than that by R(-) isomer at 30 µM. However, there was no significant difference in IC50 values or Hill coefficients between the isomers. Each point represents the mean of five to seven experiments, and error bars indicate SEM. *P < 0.05 between the isomers.

 
Percent current decay represents the fraction of the current that decays during nicotine application. Both isomers of MPPB significantly increased percent current decay at 30 and 100 µM. There was no stereoselective difference in this variable at any concentration studied. When we calculated the ratio of percent current decay in the presence of MPPB isomers to the corresponding value at control, the ratios for both isomers increased in a dose-dependent manner. There was no significant difference between the ratios for R(-) and S(+) isomer (R[-]/control and S[+]/control) at any dose (Table 1). The decaying phases of the elicited currents were well fitted to single exponential functions. Time constants of the current decay were reduced to the same extent by 30 µM R(-) and S(+)MPPB. Time constant ratios were 0.51 ± 0.1 and 0.47 ± 0.09 for R(-) and S(+) isomers, respectively. They were not significantly different from each other.


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  		Table 1. Changes in Percent Current Decay by MPPB Isomers
 
The instantaneous current-voltage relationships in the presence of the isomers at 30 µM were also similar to each other (Fig. 3). The differences in the magnitudes of inhibition by the isomers were <10% at -20, -40, or -60 mV in the tested cells. These findings indicate that the effects of MPPB are not stereoselective at the membrane potentials studied.



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  		Figure 3. The current-voltage relationship of the nicotine-induced current in the absence and presence of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) isomers. Instantaneous current-voltage curves were obtained for nicotine 30 µM alone (nicotine alone) and for nicotine with 30 µM R(-) or S(+)MPPB. A ramp pulse of 30 to -70 mV (100 mV/200 ms) was applied to a cell every 200 ms, and current traces near the peak current were subtracted from those in the absence of agonist. Three cells that exhibited slow desensitization were chosen for this experiment to avoid a large decline of the current during the ramp pulse. R(-) and S(+) isomers depressed the inward current to a similar extent at the membrane potentials studied. Similar current-voltage curves were obtained with two other cells.

 
CHEB, another convulsant barbiturate, also inhibited the nicotine-induced inward current at -60 mV. The characteristics of blockade were similar to those of MPPB's blockade at 10 and 100 µM; i.e., the inhibition was reversible, dose-dependent, and greater on the nondesensitized current than on the peak current (Fig. 4).



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  		Figure 4. Inhibition of the nicotine-induced current by 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid (CHEB). CHEB 10 or 100 µM was co-applied with nicotine 30 µM without preincubation. The nicotine-induced current in the presence of CHEB was normalized to the average of the control responses before and after CHEB. CHEB dose-dependently suppressed the nicotine-induced current. At both doses, inhibitory effects were greater on the nondesensitized current than on the peak current. Each column represents the mean ± SEM from six experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
All three barbiturates, one depressant and two convulsant, inhibited the nicotine-induced current in PC12 cells. We previously reported that thiopental, another anesthetic barbiturate, similarly inhibited the nicotine-induced current (7). The inhibitory actions of MPPB isomers on neuronal nAchRs were almost the same in their potency, despite their opposite behavioral effects. These results suggest that there is no correlation between sedative or depressive effects by the barbiturates on the CNS and the inhibitory actions on ganglionic nAchRs. This finding strongly opposes the idea that the inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates.

The depressant isomer of MPPB potentiates, but the convulsant isomer inhibits, the GABA-induced Cl flux in mouse brain membrane, reflecting their in vivo effects on the CNS (17). We have also confirmed that the isomers exhibit the opposite effects on the GABA-induced current in cultured cortical neurons (unpublished data). These findings are consistent with the view that modulation of GABAA receptors is important for barbiturate anesthesia. The modulatory effects of MPPB on GABAA receptors were observed at concentrations >=3 µM. However, potentiation in Cl flux by R(-)MPPB at 30 µM was less than that by pentobarbital at the 50% effective concentration for inducing anesthesia (50 µM) (17). In the present study, MPPB isomers significantly depressed the nondesensitized component of the nicotine-induced current at 10 µM (Fig. 2B), which suggests that MPPB isomers are very potent in inhibiting neuronal nAchRs. Thiopental also inhibits the nondesensitized current at concentrations less than the 50% effective concentration for general anesthesia in the same preparation (7). These observations suggest that neuronal nAchRs are sensitive to these barbiturates at concentrations lower than those relevant for general anesthesia.

The lack of streoselectivity of the inhibition by MPPB isomers indicates that the site(s) of action of these compounds does not discriminate the chiral structure of MPPB. One of the possible target sites would be the lipid component of plasma membranes, which reportedly does not discriminate stereoisomers (18,19). Perturbation of membrane lipids by binding of MPPB isomers may induce conformational changes in neuronal nAchRs, leading to an alteration in their functions, but the lack of stereoselectivity does not necessarily rule out the receptor protein as the potential site of action. Because MPPB isomers accelerated the current decay, they probably inhibited the current by an open channel mechanism or augmentation of desensitization (20). It is possible that MPPB molecules bind to the protein or the interface between the protein and boundary lipid at a site remote from the chiral center to exert their nonstereoselective actions.

CHEB is also a convulsive barbiturate, and the experiments using CHEB confirmed that inhibition of neuronal nAchRs by convulsant barbiturates is not specific for S(+)MPPB. The lack of correlation between anesthetic effects of barbiturates and the inhibition of neuronal nAchRs is in agreement with an earlier study (21) reporting that pentobarbital enantiomers exhibit very weak stereoselectivity in inhibition of muscle-type nicotinic receptors derived from Torpedo electroplaques. Despite a significant difference in their potency as anesthetics, the IC50 values of the enantiomers for inhibiting the carbachol-induced cation flux were very similar to each other in that preparation, which may reflect a common feature of the sites of barbiturates' action in the nicotinic receptors from Torpedo electroplaques and PC12 cells. Extensive homology of the amino acid sequence between these receptors has been identified, especially in the M2 regions forming the channel pore (1). The barbiturates may act on the ion channel pore to exert an open channel block, and this site may not discriminate the stereoisomers with different anesthetic potency. Further studies are required to clarify this hypothesis.

In contrast, isoflurane exhibits stereoselective inhibition of neuronal nAchRs from molluscan CNS according to their anesthetic potency (18). However, molluscan receptors have biophysical characteristics different from mammalian neuronal nAchRs; i.e., the former have an extraordinary high affinity to acetylcholine and are highly permeable to Cl instead of monovalent cations (5). These different properties of molluscan receptors, as well as different mechanisms of the action of isoflurane, may account for the different results.

The similar effects of the barbiturates on muscle-type and neuronal-type receptors suggest that the barbiturates studied in this experiment may exert similar actions on nicotinic receptors in the CNS through a common mechanism. However, neuronal nAchRs in the CNS are heterogeneous and have subunit combinations and pharmacological properties different from ganglionic type receptors (2,22). Central and ganglionic receptors are formed from the pentameric arrangement of one or more individual subunits (2). Whereas PC12 cells express multiple subunits of neuronal nAchRs, including {alpha}3, {alpha}5, {alpha}7, ß2, ß3, and ß4 subunits (12,23), central-type receptors consist of multiple subtypes formed with {alpha}2–9 and ß2–4 subunits. The dominant subtypes are considered to be {alpha}3-containing receptors for PC12 cells and {alpha}4ß2 receptors for the brain (2,22). {alpha}7 Homomeric receptors have pharmacological and biophysical characteristics very different from other heteromeric receptors composed with {alpha}- and ß- class subunits (2,22). However, it is unlikely that {alpha}7 receptors contribute to the current observed in our study because of their low affinity to nicotinic agonists and fast decay kinetics (24). Neuronal nAchRs in PC12 cells exhibit electrophysiological profiles similar to those of the recombinant {alpha}4ß2 receptors: both subtypes have high calcium permeability and the strong inward rectifying characteristics in the current-voltage relationships (2,25–27). Regarding agonist and antagonist pharmacology, there are differences in the rank order of sensitivities to agonists and antagonists between these receptors (23,27–30). Therefore, we cannot automatically extend the results obtained from ganglionic-type receptors in the present experiments to the receptors in the CNS.


   Acknowledgments
 
This work was supported in part by grant-in-aid for scientific research (08671761 to TA and 08771216 to RF) from the Ministry of Education, Science and Culture, Japan.

We are grateful to Prof. J. Knabe for providing the stereoisomers of MPPB and to Prof. F. Okumura for reviewing this work.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication February 24, 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