Departments of Anesthesiology and Pharmacology, Box 50, LC-203A, Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021, USA
Corresponding author. E-mail: hchemmi@med.cornell.edu
Accepted for publication: October 7, 2002
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Abstract |
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Methods. Synaptosomes or membranes prepared from rat cerebral cortex were used to analyse drug effects on [35S]t-butyl bicyclophosphorothionate ([35S]TBPS) binding to the picrotoxinin site of GABAA receptors, [3H]batrachotoxinin A 20- benzoate ([3H]BTX-B) binding to site 2 of voltage-gated Na+ channels, (+)-[methyl-3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarboxyl-2,6-dimethyl-3-pyridinecarboxylate ([3H]PN200-110; isradipine) binding to L-type Ca2+ channels, and [cyclohexyl-2,3-3H](N)glibenclamide ([3H]GB) binding to KATP channels.
Results. I.V. anaesthetics other than ketamine preferentially inhibited [35S]TBPS binding (etomidate alphaxalone > propofol > thiopental > pentobarbital). Volatile anaesthetics inhibited both [35S]TBPS and [3H]BTX-B binding with comparable potencies (halothane
isoflurane
enflurane). Antiepileptic drugs preferentially antagonized either [35S]TBPS (diazepam > phenobarbital) or [3H]BTX-B (phenytoin > carbamazepine) binding. Local anaesthetics (lidocaine, tertracaine) selectively antagonized [3H]BTX-B binding. None of the drugs tested were potent antagonists of [3H]PN200-110 or [3H]GB binding.
Conclusions. Comparative radioligand binding assays identified distinct classes of general anaesthetic and antiepileptic drugs based on their relative specificities for a defined target set. I.V. anaesthetics interacted preferentially with GABAA receptors, while volatile anaesthetics were essentially equipotent at Na+ channels and GABAA receptors. Antiepileptic drugs could be classified by preferential actions at either Na+ channels or GABAA receptors. Anaesthetics and antiepileptic drugs have agent-specific effects on radioligand binding. Both general anaesthetics and antiepileptic drugs interact with Na+ channels and GABAA receptors at therapeutic concentrations, in most cases with little selectivity.
Br J Anaesth 2003; 90: 199211
Keywords: anticonvulsants; epilepsy; nerve, synaptosome
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Introduction |
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Methods |
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[3H]Batrachotoxinin A 20- benzoate ([3H]BTX-B), (+)-[methyl-3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarboxyl-2,6-dimethyl-3-pyridinecarboxylate ([3H]PN200-110; isradipine), [cyclohexyl-2,3-3H] (N)glibenclamide ([3H]GB), and [35S]t-butyl bicyclophosphorothionate ([35S]TBPS) were obtained from DuPont-New England Nuclear (Boston, MA, USA). Percoll density gradient medium was obtained from Phamacia (Uppsala, Sweden). Tetrodotoxin, scorpion venom (Leiurus quinquestriatus), bovine serum albumin (essentially fatty acid-free), alphaxalone, phenobarbital, pentobarbital, thiopental, carbamazepine, dihydrophenytoin, tetracaine, and lidocaine were obtained from Sigma Chemical Co. (St Louis, MO, USA). (+)-Etomidate was from Janssen Pharmaceuticals (Belgium), propofol from Aldrich Chemical Co. (St Louis, MO, USA), thymol-free halothane from Halocarbon Products (North Augusta, SC, USA), and isoflurane and enflurane from Abbott Laboratories (North Chicago, IL, USA). CHEB [5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid] was a gift of Dr Hall Downes (Oregon Health Sciences Center, Portland, OR, USA).
Isolation of synaptosomes
Synaptosomes, a subcellular fraction that consists of pinched-off nerve terminals, retain a number of the functional properties of intact nerve endings, including ligand binding to Na+ channels13 and L-type Ca2+ channels.14 Presynaptic localization of KATP+ channels has been demonstrated by neurotransmitter release studies.15 As GABAA receptors are predominantly postsynaptic and endogenous synaptosomal GABA inhibits [35S]TBPS binding,16 well-washed cortical membranes were used for analysis of [35S]TBPS binding to GABAA receptors. Synaptosomes were prepared by the discontinuous Percoll density gradient method of Dunkley and colleagues17 from cerebral cortex adult male SpragueDawley rats (150200 g). The synaptosomal fraction was collected from the 23%/10% Percoll interface and diluted with five volumes of assay buffer (as described below for each method). Synaptosomes were centrifuged at 23 000 g for 10 min and resuspended in the appropriate buffer to remove Percoll. Their protein concentration was determined by the method of Bradford18 using bovine serum albumin as a standard.
Cortical membrane preparation
Rat cerebral cortex was homogenized in 50 volumes of 50 mM Triscitrate buffer, pH 7.5, with a Brinkmann Polytron (Brinkmann Instruments, Westbury, NY, USA). The homogenate was centrifuged for 15 min at 20 000 g at 4°C, and the resulting pellets were resuspended in 50 volumes of 50 mM TrisHCl buffer, pH 7.5. This centrifugation and resuspension was repeated five times.19 After the final centrifugation, pellets were resuspended in the same buffer and stored at 70°C for up to 1 week.
Equilibrium binding assays
Assays were performed under conditions optimal for each particular ligand both to facilitate comparisons of our data with published control values and to provide optimal conditions for each assay.
[3H]BTX-B binding
BTX-B binds to allosteric site 2 on voltage-gated Na+ channels with an equilibrium dissociation constant (KD) of 82 nM.13 [3H]BTX-B binding to intact synaptosomes was determined in the presence of scorpion venom, which allosterically enhances [3H]BTX-B binding to Na+ channels about 16-fold without affecting non-specific binding.13 20 Binding assays were carried out in assay buffer 1 (130 mM choline chloride, 50 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH 7.4, 5.5 mM D-glucose, 0.8 mM MgCl2, 5.4 mM KCl) containing 1 mg ml1 of bovine serum albumin, 1 µM tetrodotoxin, 80 µg ml1 of scorpion venom, and 10 nM [3H]BTX-B (final concentrations), with or without test drugs, in a final volume of 0.25 ml. Binding reactions were started by addition of synaptosomes (200 µg of protein) in 0.1 ml of assay buffer 1. After incubation at 37°C for 60 min, the reaction was terminated by addition of 3 ml of ice-cold wash buffer 1 (163 mM choline chloride, 5 mM HEPESTris, pH 7.4, 1.8 mM CaCl2, 0.8 mM MgCl2). Synaptosomes were collected immediately on to Whatman GF/C glass fibre filters (Whatman, Maidstone, UK) with a Brandel cell harvester (Brandel, Gaithersburg, MD, USA) and washed twice with 4 ml of wash buffer 1. Radioactivity on the filters was determined by liquid scintillation spectrometry in Bio-Safe NA scintillation cocktail (Research Products International, Mount Prospect, IL, USA). Non-specific binding (1020% of total binding) was determined in the presence of 0.3 mM veratridine, which binds to the same site as BTX-B.13 Specific binding of 10 nM [3H]BTX-B was 0.75 (0.06) pmol mg1 protein.
[3H]PN200-110 binding
PN200-110 (isradipine) binds to a single class of binding site (dihydropyridine site) of L-type Ca2+ channels with a KD of 70 nM.14 Synaptosomes (200 µg of protein in 0.1 ml) were incubated with 0.1 nM [3H]PN200-110 in assay buffer 1 (130 mM choline chloride, 50 mM HEPES, pH 7.4, 5.5 mM D-glucose, 0.8 mM MgCl2, 5.4 mM KCl), with or without test drugs, in a final volume of 1 ml at 25°C for 60 min.14 Binding was terminated by the addition of 3 ml of ice-cold wash buffer 1 followed by immediate filtration as above. Filters were washed twice with 3 ml of wash buffer 1, and bound [3H]PN200-110 was determined by liquid scintillation spectrometry as above. Non-specific binding (1015% of total binding) was measured in the presence of 1 µM nifedipine. Initial studies revealed specific, saturable, and time-dependent binding of [3H]PN200-110; maximal binding was reached by 60 min under the conditions used. The specific binding of 0.1 nM [3H]PN200-110 was 0.18 (0.03) pmol mg1 protein. In separate experiments, the effects of propofol on [3H]PN200-110 binding to lysed synaptosomal membranes was analysed. Synaptosomal membranes (200 µg of protein in 0.1 ml) were incubated with 0.1 nM [3H]PN200-110 in assay buffer 2 (50 mM TrisHCl, pH 7.5) with or without test drugs in a final volume of 1 ml at 25°C for 60 min.21 Binding was terminated by the addition of 3 ml of ice-cold wash buffer 2 (5 mM TrisHCl, pH 7.5); the rest of the procedure followed that used in the synaptosome assays. No significant differences were observed in the effects of propofol on [3H]PN200-110 binding to synaptic membranes compared to its effects on binding to intact synaptosomes (data not shown).
[35S]TBPS binding
[35S]TBPS is a cage convulsant that binds to rat cortical membranes at a single site on GABAA receptors (the picrotoxinin site) with an apparent KD of 25 nM.22 Washed rat cerebrocortical membranes (100 µg of protein in 0.1 ml) were incubated with 5 nM [35S]TBPS, with or without test drugs, in a final volume of 0.25 ml of assay buffer 3 (50 mM Triscitrate, pH 7.5, 200 mM NaCl) at 25°C for 2 h.19 Reactions were terminated by rapid filtration with two 4 ml washes of cold assay buffer 3, and radioactivity was determined by liquid scintillation spectrometry as above. Non-specific binding (<5% of the total binding) was measured in the presence of 10 µM picrotoxin. Specific binding of 5 nM [35S]TBPS was 3.7 (0.32) pmol mg1 protein.
[3H]Glibenclamide binding
[3H]GB binds to a single class of binding site on KATP channels with a KD of 100440 pM in rat cortical membranes.23 Synaptosomes (200 µg of protein in 0.1 ml) were incubated with 100 pM [3H]GB with or without test drugs in a final volume of 0.5 ml of assay buffer 4 (50 mM TrisHCl, pH 7.2) at 37°C for 30 min. Incubations were terminated by rapid filtration followed by three washes with 3 ml of ice-cold assay buffer 3, and bound radioactivity was determined by liquid scintillation spectrometry. Non-specific binding (1015% of total binding) was determined in the presence of 100 nM glibenclamide. The specific binding of 100 pM [3H]GB was 9.1 (1.3) fmol mg1 protein.
Specific binding was determined as the difference between total and non-specific binding. None of the drugs used affected non-specific binding (data not shown).
Drug preparation
Efforts were made to analyse concentrations of drugs that inhibited radioligand binding to 2080% of control; however, in some instances maximal drug concentration was limited by agent solubility. Stock solutions of propofol, thiopental, etomidate, alphaxalone, carbamazepine, phenytoin, phenobarbital, and CHEB were prepared in dimethyl sulphoxide, stored at 20°C, and diluted in corresponding assay buffers on the day of experiment. Dimethyl sulphoxide at 0.051% (v/v) did not affect any of the measured variables (data not shown). Stock solutions of pentobarbital, ketamine, tetracaine, and lidocaine were made in the respective assay buffers. Volatile anaesthetics were tested both as diluted solutions in dimethyl sulphoxide and as saturated buffer solutions, with similar effects. Assays with volatile anaesthetics were performed in capped tubes; aqueous concentrations in parallel assays were measured by gas chromatography.24 Reported drug concentrations refer to total in the assay concentrations. These provide an upper limit of the free concentrations, which may be considerably lower for drugs with substantial protein/lipid binding.
Statistical analysis
Results are reported as mean (SD). Statistical differences between control and experimental values were determined by analysis of variance (ANOVA) with Fishers post hoc test. Binding data were fitted to a single-site sigmoidal concentrationeffect curve with variable slope (Y=[L]h([L]hIC50h)), where Y is per cent of bound radioligand, [L] is drug concentration, IC50 is the drug concentration at half maximal inhibition, and h is the slope or Hill coefficient) by non-linear regression using GraphPad Prism v. 3.02 (GraphPad Software, San Diego, CA, USA). Curves were fit to Emax=100% inhibition; goodness of fit (R2) was >0.90 for all curves except that for CHEB effects on [3H]BTX-B binding. Each experiment included two to three replicates for each data point and was performed three to five times.
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Results |
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The volatile anaesthetics were all effective inhibitors of [35S]TBPS binding (IC50 values 0.91.2 mM). The Hill slopes for these agents were 2 (1.62.6).
[3H]BTX-B binding
Of the i.v. agents tested, propofol and ketamine inhibited [3H]BTX-B binding most potently (Fig. 1, Table 1); alphaxalone, etomidate, and pentobarbital were considerably less potent. Thiopental (up to 2 mM) did not significantly affect [3H]BTX-B binding (compared with control without drug). The Hill slopes for these agents were near 1 (0.661.4). Only propofol had a significant effect within its clinically relevant concentration range.
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Volatile anaesthetics were weak inhibitors of [3H]PN200-110 binding relative to their clinical concentrations (estimated IC50 values >3.5 mM).
[3H]GB binding
None of the general anaesthetics tested (propofol, etomidate, ketamine, thiopental, halothane, and isoflurane up to 1 mM; pentobarbital and enflurane up to 2 mM) affected [3H]GB binding significantly (data not shown).
Antiepileptic drugs
[35S]TBPS binding
Phenobarbital significantly inhibited [35S]TBPS binding with a Hill slope 1. Carbamazepine and phenytoin were minimally effective. Only phenobarbital was effective in its therapeutic concentration range. The convulsant barbiturate CHEB inhibited [35S]TBPS binding much more potently than that of the other radioligands tested; it was more potent in this action than the antiepileptic drugs tested.
[3H]BTX-B binding
Carbamazepine and phenytoin were considerably more potent as inhibitors of [3H]BTX-B binding than phenobarbital (Fig. 3, Table 1). Only phenytoin was clearly effective at therapeutic concentrations. The Hill slopes for these agents were 1 (0.71.3).
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[3H]GB binding
No significant effects on [3H]GB binding were observed with carbamazepine (up to 1 mM), phenytoin (up to 1 mM), or phenobarbital (up to 2 mM) (data not shown).
Local anaesthetics
[3H]BTX-B binding
Tetracaine and lidocaine inhibited [3H]BTX-B binding more selectively than the other radioligands tested (Fig. 4, Table 1). Inhibition correlated with their clinically effective concentrations in producing local anaesthesia. The Hill slopes for these agents were 1.
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[35S]TBPS binding
Tetracaine and lidocaine only weakly inhibited [35S]TBPS binding at supratherapeutic concentrations.
[3H]GB binding
Tetracaine (up to 1 mM) and lidocaine (up to 2 mM) did not significantly affect [3H]GB binding (data not shown).
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Discussion |
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Limitations
Radioligand binding is a versatile, reproducible technique that is amenable to high volume comparative analysis of multiple drug effects, but it is subject to certain limitations. A major limitation of this technique is that drug effects on radioligand binding do not necessarily reflect effects on target function, although considerable evidence supports such a correlation for many targets. Another limitation, which applies to all studies of isolated drug targets, is that the degree of effect on an isolated target in vitro may not correlate with the magnitude of the effect in vivo. Thus, effects detected in vitro may be magnified or minimized in a physiological context.
Radioligand binding results are limited in that each radioligand is sensitive to direct or allosteric interactions with a single site on the target of interest, most of which are large multimeric proteins. For example, weak or absent effects observed using a radioligand specific for one site could lead to the erroneous conclusion that no interaction exists, when a potent effect may be observed with a second radioligand to another site (as occurs with the Na+ channel25). The specificity of a radioligand for a single receptor subtype also limits the generalization of results to other related receptor subtypes. For example, weak effects on [3H]PN200-110 binding to L-type Ca2+ channels do not exclude significant effects on other Ca2+ channel subtypes. This limitation is a particular problem for K+ channels, for which few specific ligands are available. Our choice of a single species and brain region facilitated comparisons between drugs and with other studies, but could obscure important species, tissue or region-specific effects. Despite these limitations, this approach allows a useful comparison of anaesthetic and antiepileptic drugs on multiple targets in a single preparation under similar conditions.
Therapeutic drug concentrations
The clinically relevant concentrations of the study drugs (EC50 values for i.v. drugs; minimum alveolar concentration [MAC] for volatile anaesthetics) are used as benchmarks for evaluating the relevance of the observed IC50 values to their therapeutic effects (Table 1). Although a direct correlation between the two is not necessary, some overlap between the concentrationeffect curve in vitro with the clinically effective concentrations should exist. For the i.v. drugs, accurate effect site concentrations are rarely available, but must be derived from blood concentrations obtained at pseudo-steady state after corrections for lipid and protein binding. The derivation of the EC50 values for pentobarbital and thiopental has been described.9 The value for propofol is taken as twice the EC50 value for loss of the righting reflex in tadpoles.26
I.V. anaesthetics
Of the i.v. anaesthetics, the neurosteroid alphaxalone was highly selective for inhibition of [35S]TBPS binding to GABAA receptors over [3H]BTX-B binding to Na+ channels or [3H]PN200-110 binding to Ca2+ channels. The imidazole etomidate and the barbiturates pentobarbital and thiopental showed intermediate selectivity for [3H]BTX-B binding over [3H]PN200-110 binding; they also inhibited [35S]TBPS binding at their clinical concentrations. Propofol showed low selectivity for inhibition of [3H]BTX-B binding vs [35S]TBPS binding; clinical concentrations of this agent are expected to have significant effects on both GABAA receptors and Na+ channels.
The effects of i.v. anaesthetics on the interaction of [35S]TBPS with GABAA receptors have been addressed in several studies (see below), but never compared directly with their effects on other ligandreceptor interactions. Our results demonstrating neurochemical actions of most i.v. anaesthetics on [35S]TBPS binding to GABAA receptors are comparable with those of previous studies (Table 2). Some of the quantitative differences obtained with different preparations may result from variations in GABAA receptor subunit isoform composition.27
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Ketamine is an exception to the relatively high potency of i.v. anaesthetics for GABAA receptor potentiation.9 Potent inhibition of [3H]BTX-B binding by ketamine is consistent with its activity as a local anaesthetic.33 Very weak antagonism of [3H]PN200-110 binding to L-type Ca2+ channels was observed. This is consistent with the minimal myocardial depression produced by ketamine compared with other anaesthetics, for which significant myocardial depression results in part from block of myocardial Ca2+ channels. Ketamine inhibited veratridine-induced release of GABA with an IC50 of 100 µM without significantly affecting KCl-evoked release from rat brain synaptosomes,34 further supporting greater antagonism of Na+ channels than of Ca2+ channels. Antagonism of N-methyl-D-aspartate (NMDA) receptors resulting in disruption of glutamatergic transmission is thought to underlie the dissociative anaesthetic action of ketamine,35 although other actions such as blocking of neuronal nicotinic acetylcholine receptors may also be involved. The clinical profiles of other i.v. anaesthetics differ qualitatively from the dissociative anaesthesia produced by ketamine, in keeping with their distinct profiles for inhibition of radioligand binding inhibition.
A role for L-type voltage-gated Ca2+ channels in general anaesthesia has been suggested by findings that the L-type Ca2+ channel antagonist nitrendipine increases the anaesthetic potencies of ethanol, pentobarbitone, and benzodiazepines in mice.36 However, i.v. anaesthetics were weak antagonists of [3H]PN200-110 binding to the dihyropyridine site of L-type Ca2+ channels. In fact, [3H]PN200-110 was the least sensitive ligand for all agents, other than [3H]GB, tested except for the barbiturates, and none of the agents were effective at clinical concentrations. Hirota and Lambert37 also reported K50 values (IC50 values corrected for the competing mass of the radioligand) for inhibition of [3H]PN200-110 binding in rat cortical membranes for i.v. anaesthetics (Table 2), which they interpreted as consistent with small effects at clinical concentrations for all agents except ketamine. However, some of their IC50 values were estimated by extrapolation from incomplete concentrationeffect curves, and may thus be underestimates. Other studies also support the conclusion that dihyropyridine binding to brain L-type Ca2+ channels is relatively insensitive to i.v. anaesthetics (Table 2). Binding of [3H]verapamil, a phenylalkylamine which binds to the L-type Ca2+ channel 1 subunit at a site distinct from that of the dihydropyridines, was also insensitive to various i.v. anaesthetics.38 Collectively, these findings support our conclusion that L-type Ca2+ channels do not play a major role in the anaesthetic actions of i.v. anaesthetics.
Volatile anaesthetics
The volatile anaesthetic agents tested had significant effects on both [3H]BTX-B and [35S]TBPS binding at clinical concentrations; there was 2-fold greater potency in inhibition of [3H]BTX-B binding vs [35S]TBPS binding. This supports the hypothesis that these agents act through combined actions on both Na+ channels and GABAA receptors. Inhibition of CNS Na+ channels by volatile anaesthetics at clinical concentrations has also been demonstrated electrophysiologically39 and biochemically.24
Volatile anaesthetics reversibly inhibit peak current and induce a hyperpolarizing shift in the voltage dependence of steady-state inactivation of rat neuronal Na+ channels stably expressed in Chinese hamster ovary cells39 or in isolated dorsal root ganglion neurons.40 Volatile anaesthetics also inhibit veratridine-evoked glutamate release from isolated nerve terminals (halothane IC50=670 µM) with greater potency than release evoked by elevated KCl (no effect at 650 µM).3 24 This pattern of inhibition is consistent with greater sensitivity of presynaptic Na+ channels to inhibition by volatile anaesthetics than of the Ca2+ channels coupled to glutamate release. Volatile anaesthetic action at presynaptic Na+ channels is further supported by our findings that halothane inhibits veratridine-evoked 22Na+ influx and increases in intracellular [Na+] and [3H]BTX-B binding (IC50=530 µM) in rat cortical synaptosomes at concentrations achieved clinically.24
Volatile anaesthetics have well-characterized electrophysiological actions at GABAA receptors: they enhance Cl currents in response to subsaturating concentrations of GABA by increasing open channel probability.9 Neurochemical actions include enhanced 36Cl uptake into synaptoneurosomes and inhibition of [35S]TBPS binding (Table 2).19 Antagonism of [35S]TBPS binding has also been reported in mouse membrane vesicles.41 Our IC50 values for [35S]TBPS binding are consistently lower than those of Moody and colleagues,19 possibly because of differences in protein concentration or membrane preparation.
In contrast to their potent effects on [3H]BTX-B and [35S]TBPS binding, we observed <25% inhibition of [3H]PN200-110 binding at volatile anaesthetic concentrations of 1 mM. Previous studies examining the effects of volatile anaesthetics on [3H]PN200-110 binding to L-type Ca2+ channels have been inconsistent. Drenger and colleagues21 reported that halothane inhibits [3H]PN200-110 binding to rat cortical membranes (32% inhibition at 0.78 vol%; Emax=44% inhibition), while isoflurane (2.3 vol%) and enflurane (4.8 vol%) were ineffective. However, Moody and colleagues observed significant inhibition of [3H]PN200-110 binding by (+)-isoflurane using mouse cortical membranes (IC50190 µM).42 These assays are apparently markedly dependent on conditions such as radioligand concentration, membrane preparation, species, and/or incubation conditions. We employed isolated rat cerebrocortical nerve terminals, while others employed rat forebrain membranes21 or mouse cerebrocortical membranes. This suggests the possibility that nerve terminal L-type Ca2+ channels may be less sensitive to volatile anaesthetics than somatodendritic channels (the principal subcellular location of neuronal L-type Ca2+ channels43). This is supported by the observation that glutamate release from rat cerebrocortical synaptosomes evoked by elevated KCl (which activates voltage-gated Ca2+ channels) is insensitive to L-type Ca2+ channel antagonists or to volatile anaesthetics at clinical concentrations.3 In contrast, electrophysiological experiments indicate inhibition of somatic Ca2+ channels at clinical concentrations of halothane and isoflurane in cultured hippocampal pyramidal neurons (T-, L-, N- and P-type channels),44 isolated dorsal root ganglion neurons (T-, L-, and N-type channels)4547 and isolated recombinant Ca2+ channels expressed in Xenopus oocytes.48 The involvement of volatile anaesthetic inhibition of voltage-gated Ca2+ channels, and of L-type channels in particular, in the production of the anaesthetic state remains unclear,10 but appears unlikely from the present data.
Antiepileptic drugs
The mechanisms of action of currently used antiepileptic drugs2 can be broadly classified into one or more of three categories: (i) enhancement of inhibitory GABAergic transmission; (ii) reduction of excitatory glutamatergic transmission; and (iii) modulation of Na+, Ca2+, or K+ channels. Benzodiazepines and barbiturates enhance GABAergic transmission at their therapeutic free serum concentrations. The established drugs phenytoin and carbamazepine, as well as the newer agents lamotrigine and riluzole, produce use-dependent block of voltage-gated Na+ channels, a property considered integral to their efficacy in the treatment of generalized tonic-clonic seizures and some partial seizures. Benzodiazepines and barbiturates may also block Na+ channels at the high serum concentrations achieved in the treatment of status epilepticus. Our findings concerning the relative potencies in radioligand binding assays of representative antiepileptic drugs at these targets support these general conclusions. Carabamazepine and phenytoin were most potent in antagonizing [3H]BTX-B binding at Na+ channels, while phenobarbital was most potent in antagonizing [35S]TBPS binding to GABAA receptors. In contrast to the overlap between the clinical concentrations of general anaesthetics and their IC50 values for inhibition of [35S]TBPS binding, there were significant differences for those of phenobarbital, an antiepileptic drug thought to act via GABAA receptor modulation. The finding that [35S]TBPS binding is a good indicator for general anaesthetic interactions with GABAA receptors, but is an insensitive indicator of antiepileptic drug interactions, supports the existence of distinct sites for general anaesthetic and antiepileptic drug interactions with this receptor.9
Previous studies of the actions of antiepileptic drugs on [35S]TBPS binding are incomplete (Table 2). We found that phenobarbital was most potent in antagonizing [35S]TBPS binding at GABAA receptors; greater activity at GABAA receptors is consistent with its greater sedative effect. None of these agents are markedly selective for their preferred target; this lack of selectivity resembles that of the general anaesthetics, and likely underlies the many side effects associated with their use. The convulsant barbiturate CHEB was most potent in antagonizing [35S]TBPS binding. The finding that CHEB is only a weak inhibitor of [3H]PN200-110 binding suggests that its ability to increase intracellular Ca2+ and Ca2+-dependent glutamate release (EC50=14 µM) in rat cerebrocortical synaptosomes49 does not involve interaction with L-type Ca2+ channels.
Interactions of established antiepileptic drugs with voltage-gated Na+ channels in rat cortical synaptosomes reported previously are in general agreement with other results (Table 2).50 We have reported previously the selectivity of phenytoin (IC50=220 µM), carbamazepine (IC50=200 µM), lamotrigine (IC50=200 µM), and riluzole (IC50=4.8 µM) for block of presynaptic Na+ channel-dependent vs Ca2+ channel-dependent release of glutamate.51 A number of antiepileptic drugs can affect voltage-gated Ca2+ channels.2 For example, phenytoin, barbiturates, and riluzole reduce Ca2+ influx into synaptosomes and block neurotransmitter release.51 These effects only occur at concentrations above those achieved clinically, indicating that Ca2+ channels are not the principal targets for most established antiepileptic drugs. However, agents useful in the treatment of generalized absence epilepsy, such as ethosuximide, act primarily by inhibition of low voltage-activated T-type Ca2+ channels.2 The effects of antiepileptic drugs on [3H]PN200-110 binding to L-type Ca2+ channels have not been reported previously. Binding of the dihydropyridine [3H]nitrendipine to rat cortical membranes was reduced by phenobarbital (IC50=400 µM) and phenytoin (IC50=90 µM), while carbamazepine was ineffective up to 1 mM.52 It is unclear why these results differ so much from ours given that this ligand is thought to bind to the same site as [3H]PN200-110. The high potency of phenytoin in particular is inconsistent with previous studies indicating the greater relative potency of this agent at voltage-gated Na+ channels compared to Ca2+ channels.2 51
Local anaesthetics
Inhibition of voltage-gated Na+ channels and disruption of axonal conduction is the basis of the analgesic actions of local anaesthetics.53 Lidocaine and tetracaine were highly selective for inhibition of [3H]BTX-B binding in isolated rat cerebrocortical nerve terminals (Table 2). Clinically achieved concentrations of local anaesthetics are unlikely to have significant actions at the other channels tested. Both drugs tested were more effective at Na+ channels than at L-type Ca2+ channels by approximately 100-fold (Table 2). Lidocaine and tetracaine were very weak as antagonists of [35S]TBPS or [3H]GB binding.
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Acknowledgements |
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References |
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