Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na+) channels. In this study, the biochemical effects of halothane on Na+ channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na+ channels in anesthetic effects.
Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na+ channel-dependent Na+ influx (using 22Na+), changes in intrasynaptosomal [Na+] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na+ channel (by radioligand binding). The potential physiologic and functional significance of these effects was determined by measuring the effects of halothane on veratridine-evoked Na+ channel-dependent glutamate release (using enzyme-coupled spectrofluorometry).
Halothane inhibited veratridine-evoked 22Na+ influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na+] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [3H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na+ channel (IC50 = 0.53 mM). Scatchard and kinetic analysis revealed an allosteric competitive mechanism for inhibition of toxin binding. Halothane inhibited veratridine-evoked glutamate release from synaptosomes with comparable potency (IC50 = 0.67 mM).
Halothane significantly inhibited Na+ channel-mediated Na influx, increases in intrasynaptosomal [Na+] and glutamate release, and competed with neurotoxin binding to site two of the Na+ channel in synaptosomes at concentrations within its clinical range (minimum alveolar concentration, 1-2). These findings support a role for presynaptic Na+ channels as a molecular target for general anesthetic effects.
NEUROTRANSMISSION consists of action potential propagation along axons and chemical transmission across synapses, both of which involve voltage-dependent sodium channels (Na sup + channels). [1,2]Previous studies in mammalian and nonmammalian tissues have demonstrated inhibition of neuronal Na sup + channels by volatile anesthetic agents, including reduced axonal conduction, [3,4]increased firing threshold, [5,6]and altered Na sup + channel gating and conductance. [7,8]Recent evidence supports inhibition of central nervous system (CNS) Na sup + channels at clinical concentrations in cells transfected with rat brain type IIA Na sup + channels. Voltage- and use-dependent suppression of Na sup + channel currents was found at physiologic resting membrane potentials, which provides direct evidence that Na sup + channels are a sensitive molecular target for volatile anesthetic action.
Volatile anesthetic agents also inhibit release of neurotransmitters in the CNS. [3,10–14]Possible mechanisms include effects on presynaptic terminal depolarization; Ca2+ influx; and synthesis, storage, exocytosis, and inactivation of neurotransmitters. Invading action potentials depolarize the presynaptic plasma membrane by activation of Na sup + channels, which leads to Ca2+ entry through activation of voltage-dependent Ca2+ channels, followed by Ca2+-dependent exocytotic release of neurotransmitters. Inhibition of neuronal Ca2+ channels by volatile anesthetic agents has been demonstrated by effects on intracellular [Ca2+], [15,16]radioligand binding, [17,18]and Ca2+ currents. Data from our laboratory, however, suggest that a step proximal to Ca2+ influx, i.e., Na sup + influx through Na sup + channels, is more sensitive than Ca2+ influx through Ca2+ channels to the presynaptic actions of volatile anesthetic agents, although this is controversial. Peripheral neuronal Na sup + channels also appear to be more sensitive than Ca2+ channels to the action of isoflurane. 
In the current study, we analyzed the effects of the volatile anesthetic agent halothane on presynaptic CNS Na sup + channels by measuring its effects on veratridine-evoked22Na sup + influx and increases in intrasynaptosomal [Na sup +]([Na sup +]i) and on the binding of radiolabeled neurotoxins to Na sup + channels in rat cerebrocortical synaptosomes. We also examined the functional effects of halothane on presynaptic Na sup + channels by measuring veratridine-evoked release of glutamate, the major excitatory neurotransmitter in the CNS, from the same preparation. Synaptosomes, a subcellular fraction that consists of pinched-off nerve terminals, provide a useful system for analyzing the biochemical pharmacologic characteristics of presynaptic Na sup + channels. [21–24]Our results indicate an interaction between halothane and CNS Na sup + channels and that presynaptic Na sup + channels may mediate some of the inhibitory effects of volatile anesthetic agents on excitatory synaptic transmission.
Materials and Methods
Reagents were obtained from the following sources:[11-sup 3 H]saxitoxin (28 Ci [center dot] mmol sup -1) from Amersham (Arlington Heights, IL);[sup 3 H]batrachotoxinin-A 20-alpha-benzoate (BTX-B; 34 Ci [center dot] mmol sup -1) and22NaCl (1 mCi [center dot] ml sup -1) from DuPont-New England Nuclear (Boston, MA);[42-sup 3 H] brevetoxin-3 (14.25 Ci [center dot] mmol sup -1) and Ptychodiscus brevis toxin-3 (PbTx-3) from Chiral Corp. (Miami, FL); Percoll density gradient medium from Pharmacia/LKB (Uppsala, Sweden); halothane (thymol-free) from Halocarbon Products (North Augusta, SC); tetrodotoxin, veratridine, scorpion venom (Leiurus quinquestriatus), L-glutamate dehydrogenase (Proteus sp), and dimethylsulfoxide from Sigma Chemical Co. (St. Louis, MO); and the cell permeant acetoxymethyl ester precursor form of Na sup +-binding benzofuran isophthalate (SBFI-AM) and Pluronic F-127 from Molecular Probes, Inc. (Eugene, OR). All other chemicals were of reagent grade.
Dimethylsulfoxide at a final concentration of 0.05%(vol/vol) was used as a vehicle in binding and flux studies to minimize reaction volumes. Control experiments showed that the vehicle alone had no effect on the variables measured (data not shown).
Preparation of Synaptosomes
Synaptosomes from rat cerebral cortex were prepared using a modification of the procedure of Dunkley et al. Adult male (150–175 g) Sprague-Dawley rats were anesthetized with 80% CO2/20% O2, were killed by decapitation, and their brains were immediately removed and rinsed in ice-cold 0.32 M sucrose. Cortical gray matter was dissected and homogenized in ten volumes of 0.32 M sucrose using a motor-driven Teflon glass homogenizer at 900 rpm for 10 up-and-down strokes. The homogenate was centrifuged at 1,000 x g for 2 min. The supernatant was collected and centrifuged at 15,000 x g for 12 min. The resulting pellet was resuspended in 8 ml of 0.32 M sucrose. Aliquots (2 ml) of this fraction were loaded onto discontinuous gradients consisting of three 2.5-ml layers of filtered (0.45 micro meter) Percoll density gradient medium (23%, 10%, and 3%) in 0.32 M sucrose plus 0.25 mM dithiothreitol and 1 mM ethylenediaminetetraacetic acid, pH 7.4. The gradients were centrifuged at 25,000 x g for 6.5 min. The synaptosome fraction was collected from the 23%/10% Percoll interface and diluted approximately fivefold in low Na sup + buffer for22Na sup + influx studies, Na sup +-free buffer for [Na sup +]iand for neurotoxin binding studies, or high Na sup + buffer for glutamate release assays (buffer compositions given subsequently); all buffers were equilibrated with 95% O2/5% CO2. The synaptosomes were centrifuged at 23,000 x g for 10 min and resuspended in the appropriate buffer. Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard.
Measurement of sup 22 Na sup + Influx
sup 22 Na sup + influx was measured by a modification of the method of Tamkun and Catterall. Synaptosomes (600–700 micro gram protein in 150 micro liter low Na sup + buffer, consisting of 130 mM oholine chloride, 5.4 mM KCl, 5 mM NaCl, 0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM HEPES-Tris, pH 7.4) were preincubated at 37 [degree sign] Celsius for 5 min in the absence or presence of halothane (added as a diluted solution in dimethylsulfoxide). After preincubation, 60 micro meter veratridine with or without 80 micro gram/ml scorpion venom was added, and the samples were incubated for 10 min at 37 [degree sign] Celsius. Uptake was initiated by the addition of 1.3 micro Ci of carrier-free22NaCl in 50 micro liter low Na sup + buffer and was terminated after 5 s by the addition of 3 ml of ice-cold washing buffer (163 mM choline chloride, 0.8 mM MgSO4, 1.8 mM CaCl2, and 5 mM HEPES-Tris, pH 7.4) and rapid vacuum filtration through GF/C glass fiber filters (Whatman, Kent, UK). Filters were washed twice with 3 ml washing buffer, and filter radioactivity was determined by liquid scintillation spectrometry using Bio-Safe NA scintillation cocktail (Research Products International Corp., Mount Prospect, IL). Nonspecific (Na sup + channel-independent)22Na sup + uptake was determined in the presence of 1 micro meter tetrodotoxin, a specific Na sup + channel blocker. 
Measurement of Free Intrasynaptosomal [Na sup +]
Na sup + concentration was determined by ion-specific spectrofluorometry using a spectrofluorometer (Perkin Elmer LS-50B; Beaconsfield, UK) with continuous computer-assisted data acquisition. SBFI-AM was used as the fluorescent indicator. Synaptosomes (5 mg protein) were suspended in 1 ml of a Na sup +-free buffer (120 mM choline chloride, 5 mM KCl, 0.8 mM MgSO4, 5 mM D-glucose, and 50 mM HEPES-Tris, pH 7.4) containing 10 micro Meter SBFI-AM and 0.01%(vol/vol) Pluronic F-127 (a non-ionic detergent that facilitates indicator uptake) and incubated for 2 h at room temperature. At the end of the loading period, synaptosomes were centrifuged at 5,000 x g, resuspended in indicator-free buffer, and centrifuged again at 5,000 x g to remove excess indicator. The synaptosomes were suspended in Na sup +-free buffer and incubated for an additional 30 min to allow indicator hydrolysis. After incubation, aliquots of synaptosomes (0.5 mg protein) were centrifuged, and the pellets were stored on ice until use. For free [Na sup +]idetermination, synaptosome pellets were resuspended in 1.5 ml of 120 mM Na sup + buffer (same as Na sup +-free buffer, except NaCl was replaced choline chloride) and incubated in a stirred quartz cuvette at 37 [degree sign] Celsius in the absence or presence of halothane (added as aliquots of saturated buffer solution) for 5 min, followed by the addition of 60 micro Meter veratridine to activate Na sup + channels. Synaptosomal [Na sup +]iwas calculated by the fluorescence ratio method at an emission wavelength of 510 nm, with excitation wavelengths of 340 and 380 nm (switched every 2 s). The signal ratio was converted into free [Na sup +]ibased on the method of Grynkiewicz et al. Calibration of the 340:380 nm excitation ratio in terms of free [Na sup +]iwas performed for each synaptosome preparation. For calibration, SBFI-loaded synaptosomes were added to solutions of known extracellular [Na sup +] made by appropriate mixtures of high-[Na sup +] and high-potassium ([K sup +]) solutions in the presence of 40 micro Meter monensin, 2 micro Meter gramicidin, and 100 micro Meter ouabain. The high-[Na sup +] solution contained 120 mM NaCl, 2 mM EGTA, and 10 mM HEPES-Tris, pH 7.4. The high-[K sup +] solution was identical except that K sup + replaced Na sup +. In control experiments, no quenching of SBFI fluorescence by veratridine or halothane was observed in the presence of monensin, a Na sup + ionophore (data not shown).
Equilibrium Binding Assays
All reactions were performed at 37 [degree sign] Celsius in Teflon-sealed glass vials to minimize loss of halothane.
[sup 3 H]Batrachotoxinin-A 20-alpha-Benzoate Binding. [sup 3 H]BTX-B binding was determined as described by Postma and Catterall using a Na sup +-free buffer (135 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM HEPES-Tris, pH 7.4) plus 10 nM [sup 3 H]BTX-B, 1 micro Meter tetrodotoxin, 80 micro gram/ml scorpion venom, and 1 mg/ml bovine serum albumin. Tetrodotoxin inhibits membrane depolarization due to Na sup + flux through Na sup + channels activated by BTX-B and scorpion venom. Binding reactions were initiated by rapid mixing of synaptosomes (200 micro gram protein in 100 micro liter) with 150 micro liter of the reaction mixture just described in the absence or presence of halothane (added as a diluted solution in dimethylsulfoxide) and were terminated after 60 min at 37 [degree sign] Celsius by the addition of 3 ml of ice-cold washing buffer. Synaptosomes were collected on GF/C glass fiber filters (Brandel, Gaithersburg, MD) by vacuum filtration and washed three times with 3 ml washing buffer. Bound [sup 3 H]BTX-B was determined by liquid scintillation spectrometry. Nonspecific binding (10–20% of total binding) was determined in the presence of 0.3 mM veratridine, which binds at the same site as BTX-B. 
[sup 3 H]Brevetoxin-3 Binding. [sup 3 H]Brevetoxin-3 binding was determined as described by Edwards et al., with minor modifications. Synaptosomes (100 micro gram protein in 100 micro liter) were suspended in a Na sup +-free buffer (135 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, 50 mM HEPES-Tris, pH 7.4) plus 25 nM [sup 3 H]brevetoxin-3 and 0.01%(vol/vol) Plutonic F-127, a nonionic detergent required to solubilize the high concentrations of unlabeled brevetoxin-3 used to determine nonspecific binding, in the absence or presence of halothane (added as a diluted solution in dimethyl-sulfoxide). After rapid mixing, synaptosomes were incubated at 4 [degree sign] Celsius for 1 h, after which the reaction was stopped by the addition of 3 ml ice-cold washing buffer. The synaptosomes were collected on GF/C glass fiber filters under vacuum and washed twice with 3 ml washing buffer. Bound [sup 3 H]brevetoxin-3 was determined by liquid scintillation spectrometry. Nonspecific binding (10–15% of total binding) was measured in the presence of 10 micro Meter unlabeled brevetoxin-3.
[sup 3 H]Saxitoxin Binding. [sup 3 H]Saxitoxin binding was determined as described by Catterall et al. Synaptosomes (100 micro gram protein in 100 micro liter) were added to a reaction mixture (100 micro liter) consisting of a Na sup +-free buffer (135 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM HEPES-Tris, pH 7.4) plus 3 nM [sup 3 H]saxitoxin, in the absence or presence of halothane (added as a diluted solution in dimethylsulfoxide). Samples were rapidly mixed and incubated at 37 [degree sign] Celsius for 30 min. Binding reactions were stopped by the addition of 3 ml of ice-cold washing buffer, and synaptosomes were collected on GF/C glass fiber filters under vacuum and washed twice over 10–15 s. Bound [sup 3 H]saxitoxin was determined by liquid scintillation spectrometry. Nonspecific binding (10–15% of total binding) was determined in the presence of 1 micro Meter tetrodotoxin, which binds at the same site as saxitoxin.
Kinetic Binding Assays
The time course of [sup 3 H]BTX-B dissociation from the Na sup + channel receptor complex was analyzed by preincubating synaptosomes for 60 min with 10 nM [sup 3 H]BTX-B, 80 micro gram/ml scorpion venom, and 1 micro Meter tetrodotoxin at 37 [degree sign] Celsius, as in the equilibrium binding assays. Dissociation was initiated by adding 0.3 mM veratridine in the absence or presence of halothane (0.74 mM; minimum alveolar concentration [MAC][nearly =] 2; added as a diluted solution in dimethylsulfoxide). Reactions were terminated (after 5, 10, 20, or 30 min) by vacuum filtration and washing, followed by determination of bound [sup 3 H]BTX-B. The dissociation rate constant (k sub -1) was calculated using the equation ln(Bt/Bo)=-k sub -1 [center dot] t, where Btis the specific binding of [sup 3 H]BTX-B at time t, and Bois the specific binding of [sup 3 H]BTX-B at time zero. A plot of ln(Bt/B sub o) versus t, in the absence or presence of halothane, was linear with a slope of k sub -1.
The rate of association of [sup 3 H]BTX-B was measured by incubating synaptosomes with 80 micro gram/ml scorpion venom for 15 min at 37 [degree sign] Celsius in the absence or presence of halothane (0.74 mM; added as a diluted solution in dimethylsulfoxide) as in the equilibrium binding assays. [sup 3 H]BTX-B (10 nm) was then added to the synaptosomes to initiate binding. Parallel assays were performed in the presence of 0.3 mM veratridine to determine nonspecific binding at each time point. Incubations were terminated (after 5, 10, 20, or 30 min) by vacuum filtration and washing followed by determination of bound [sup 3 H]BTX-B. The association rate constant (k sub +1) of [sup 3 H]BTX-B binding was calculated using the equation ln(Beq/Beq- Bt)=([L]k sub +1 + k sub -1)t, where Beqis the specific binding of [sup 3 H]BTX-B at equilibrium, Btis the specific binding of [sup 3 H]BTX-B at time t, [L] is the concentration of [sup 3 H]BTX-B, and k sub -1 is the dissociation rate constant for [sup 3 H]BTX-B from the Na sup + channel receptor complex at the ambient drug concentration. A plot of ln(Beq/Beq- Bt) versus t was linear, with a slope of [L]k sub +1 + k sub -1, from which k + 1 was calculated. The inhibition constant, Ki, for halothane was calculated from the equation: Ki= IC50/(1 + L/Kd), where L is the concentration of [sup 3 H]BTX-B (10 nm), Kdis the equilibrium dissociation constant for [sup 3 H]BTX-B (122 nM), and IC50is the concentration of halothane which produced 50% inhibition of [sup 3 H]BTX-B binding. The dissociation constant Kdalso was calculated from kinetic data as k sub -1 /k sub +1.
Measurement of Release of Glutamate from Synaptosomes
Endogenous release of glutamate was measured by the method of Nicholls et al. Synaptosomal pellets (0.5 mg protein) were resuspended in 1.5 ml release buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl sub 2, 1.2 mM Na2HPO4, 5 mM NaHCO3, 10 mM D-glucose, and 20 mM HEPES, pH 7.4 with NaOH) plus 16 micro Meter bovine serum albumin (essentially free of fatty acid), 1 mM NADP sup +, 100 U L-glutamate dehydrogenase, and 1.3 mM CaCl2. Stirred samples were equilibrated at 37 [degree sign] Celsius for 4 min in a spectrofluorometer cuvette, and data acquisition was started with an excitation wavelength at 340 nm and an emission wavelength at 510 nm. After recording basal glutamate release, 50–350 micro liter buffer solution saturated with halothane was added, and the rate of release of glutamate (from 0–60 s) was measured. After recording release for 200 s after the addition of halothane, veratridine (60 micro Meter) was added, and once again the rate of release of glutamate (from 0–60 s) was measured. The fluorescence signal was calibrated by adding 5 nmol L-glutamate to the cuvette at the end of each experiment.
Volatile Anesthetic Quantification
Volatile anesthetic agents were added as aliquots of saturated buffer solutions for measurement of [Na sup +]iand release of glutamate and were diluted in dimethylsuffoxide for measurement of22Na sup + influx and neurotoxin binding. Final anesthetic concentrations in each assay mixture were determined by gas chromatography. A fixed amount of the assay mixture was withdrawn from the tube/cuvette with a gas-tight syringe and extracted into n-heptane. The n-heptane extract was injected onto a gas chromatograph (GC-8A; Shimadzu Corp., Kyoto, Japan) equipped with a thermal conductivity detector. Separation was achieved on a 1.8-m-long, 6-mm ID glass column packed with Porapak Q (Supelco, Bellefonte, PA). The column temperature was 210 [degree sign] Celsius, the injector temperature was 230 [degree sign] Celsius, and carrier gas flow was 40 ml/min.
Statistical differences between control and experimental values were determined by analysis of variance with Fisher's post hoc test. Concentration-effect data were analyzed using a graded dose-response program that performs linear regression analysis on data between 20% and 80% of the maximal response (Pharm/PCS Pharmacologic Calculation System, Version 4.2; Springer Verlag, New York, NY). Confidence limits follow the derived IC50values in the text. Kdvalues and maximum number of binding sites (Bmax) for [sup 3 H]BTX-B were calculated from Scatchard plots using Enzfit (Elsevier-Biosoft, Cambridge, UK). Kinetic (association and dissociation) parameters were estimated by linear regression using the Pharm/PCS Pharmacologic Calculation System. Curves were fit to data by simple polynomial (Figure 1, Figure 2, Figure 3, Figure 6, and Figure 8) or linear (Figure 4, Figure 5(A, B), and Figure 9) functions using Origin software (Microcal Software, Inc., Northampton, MA). Each experiment contained two to three replicates for each data point and was performed n times, as specified. Values are expressed as mean +/- SD.
Experiments were performed in accordance with the National Institutes of Health guide for the care and use of laboratory animals as approved by the Cornell University Medical College Institutional Animal Care and Use Committee.
Na sup + Influx
Veratridine, an alkaloid neurotoxin that causes persistent activation of Na sup + channels by binding to site two, was used to stimulate22Na sup + influx into synaptosomes. Uptake was linear from 2–10 s (data not shown); a 5-s uptake period was used in subsequent experiments. Specific22Na sup + uptake stimulated by 60 micro Meter veratridine, which causes maximal stimulation of22Na sup + influx into rat cortical synaptosomes, was 73 +/- 6 nmol [center dot] mg sup -1 [center dot] min sup -1 (n = 3). Veratridine-evoked22Na sup + uptake was completely inhibited by 1 micro meter tetrodotoxin, indicating that it is Na sup + channel mediated. Some experiments included Leirus quin-questriatus scorpion venom, which contains alpha-scorpion toxins, small polypeptide neurotoxins that bind to site three to inhibit Na sup + channel inactivation and that interact cooperatively with toxin binding to site two. Scorpion venom augmented veratridine-evoked uptake to 138 +/- 9 nmol [center dot] mg sup -1 [center dot] min sup -1 (n = 3). Halothane inhibited veratridine-evoked22Na sup + uptake in a concentration-dependent manner in the absence (IC50= 1.1 mM; range, 0.8–1.4 mM) or presence (IC50= 1.1 mM; range, 0.8–1.4 mM) of scorpion venom with comparable efficacy and potency (Figure 1). Basal uptake (in the absence of veratridine) was not affected by halothane (data not shown).
Intrasynaptosomal [Na sup +]
Veratridine (60 micro meter) increased free [Na sup +]iin synaptosomes fourfold, from 17 +/- 2 to 67 +/- 5 mM, in 120 mM Na sup +-containing buffer (n = 4); this effect was completely blocked by 1 micro meter tetrodotoxin (data not shown). Halothane did not affect resting [Na sup +]ibut significantly inhibited veratridine-evoked increase in [Na sup +]iin a concentration-dependent manner (IC50= 0.97 mM; range, 0.6–1.4 mM; n = 3;Figure 2).
[sup 3 H]Batrachotoxinin-A 20-alpha-Benzoate Binding
[sup 3 H] BTX-B binding nearly 16-fold, from 24 +/- 3 to 380 +/- 55 fmol/mg (n = 6), without affecting nonspecific binding. Scatchard analysis of [sup 3 H]BTX-B binding in the absence of halothane revealed binding to a single class of high affinity binding sites with a Kdvalue of 122 +/- 8 nm and a Bmax of 2.43 +/- 0.18 pmol [center dot] mg protein sup -1 (n = 3;Figure 3), values which are similar to those reported previously. [23,29,36]Halothane inhibited specific [sup 3 H]BTX-B binding in a concentration-dependent manner (IC50= 0.53 mM; range, 0.37–0.71 mM; n = 3;Figure 4) without affecting nonspecific binding (data not shown); the calculated Kivalue for halothane was 0.49 mM. Halothane (0.74 mM) increased the Kdvalue for [sup 3 H]BTX-B to 478 +/- 60 nm (P < 0.05) without significantly affecting Bmax(3.14 +/- 0.43 pmol [center dot] mg protein sup -1; P = 0.14; n = 3). This effect is most consistent with a competitive mechanism for inhibition of [sup 3 H]BTX-B binding. The Hill coefficient for inhibition of [sup 3 H]BTX-B binding by halothane was 1.0 (data not shown), suggesting an interaction with a single class of binding sites.
Kinetics of [sup 3 H]Batrachotoxinin-A 20-alpha-Benzoate Binding
The k sub +1 value for [sup 3 H]BTX-B binding in the absence of halothane was 0.0032 +/- 0.0002 min sup -1 (Figure 5(A). Halothane (0.74 mM) decreased the rate of association of [sup 3 H]BTX-B to 0.0023 +/- 0.0002 min sup -1 (P < 0.05; n = 3).
The k sub -1 value for [sup 3 H]BTX-B dissociation from the Na sup + channel receptor complex was 0.005 +/- 0.001 min sup -1 (Figure 5(B)). Halothane (0.74 mM) significantly enhanced the dissociation rate to 0.010 +/- 0.002 min sup -1 (P < 0.05; n = 3). The calculated Kdvalues (Kd= k sub -1 /k sub +1) for [sup 3 H]BTX-B [sup 3 H]BTX-B binding to synaptosomes was measured in the presence of scorpion venom, which enhanced specific [sup 3 H]BTX-B were 1.54 +/- 0.23 nm in the absence and 4.35 +/- 0.5 nm in the presence of halothane (P < 0.05; n = 3). Lower Kdvalues calculated from the kinetic data compared with those obtained from equilibrium binding also were observed for [sup 3 H]BTX-B binding in cardiac myocytes. The halothane-induced increase in Kdcalculated from equilibrium binding (3.9-fold) is in good agreement with the increase calculated from the kinetic data (2.8-fold).
An effect of halothane on rebinding of dissociated [sup 3 H]-BTX-B is unlikely, because dissociation assays contained a saturating concentration of veratridine (0.3 mM), a direct competitive inhibitor of [sup 3 H]-BTX-B binding. 
[sup 3 H]Saxitoxin and [sup 3 H]Brevetoxin-3 Binding
Specific binding of [sup 3 H]saxitoxin to site one of the Na sup + channel in synaptosomes was 102 +/- 5 fmol [center dot] mg protein sup -1 (n = 3). Halothane (up to 1.2 mM) had no significant effect on [sup 3 H] saxitoxin binding (Figure 6). Halothane at 1.8 mM marginally but significantly enhanced binding (to 119 +/- 3 [center dot] mg protein sup -1; P < 0.05; n = 3). Specific binding of [sup 3 H]brevetoxin-3 to site five of the Na sup + channel in synaptosomes was 2.11 +/- 0.02 pmol [center dot] mg protein-1 (n = 3). Halothane slightly enhanced binding at concentrations of 0.74 and 1.2 mM, whereas at 1.8 mM it decreased binding.
Veratridine-evoked Release of Glutamate
Veratridine-evoked release of glutamate is Na sup + channel-dependent (tetrodotoxin-sensitive)and therefore can be used to assess the functional significance of presynaptic Na sup + channel inhibition by halothane. Veratridine-evoked release of glutamate in the presence of 1.3 mM Ca2+ was completely blocked by 1 micro meter tetrodotoxin (data not shown). Halothane inhibited veratridine-evoked release of glutamate in a concentration-dependent manner (IC50= 0.67 mM; range, 0.57–0.89 mM; n = 3;Figure 7and Figure 8). Correlations between the percentage inhibition by halothane of release of glutamate versus22Na sup + influx (r2= 0.94), change in [Na sup +]i(r2= 0.99), or inhibition of [sup 3 H]BTX-B binding (r2= 0.96) were essentially linear (Figure 9). The data for release of glutamate versus22Na sup + influx and [Na sup +] showed comparable slopes and intercepts, whereas the datafor [sup 3 H]-BTX-B binding showed a more shallow slope and a positive y intercept.
Indirect electrophysiologic evidence suggests that volatile anesthetic agents inhibit excitatory synaptic transmission by a presynaptic mechanism. [13,14]Previous evidence from our laboratory has implicated blockade of presynaptic Na sup + channels in the inhibition of release of glutamate from rat cortical synaptosomes by volatile anesthetic agents. The results of the current study provide additional evidence that halothane alters nerve terminal function by interacting with Na sup + channels to inhibit release of glutamate.
Halothane inhibited veratridine-evoked22Na sup + influx into synaptosomes. Because22Na sup + influx is directly proportional to ion channel Na sup + permeability and to the number of open channels, reductions in Na sup + influx reflect reduced presynaptic Na sup + channel opening or permeability. Halothane did not affect22Na sup + influx into synaptosomes in the presence of tetrodotoxin, which indicates a lack of effects on other modes of Na sup + entry.
Inhibition by halothane of22Na sup + influx through presynaptic Na sup + channels was confirmed by its inhibition of veratridine-evoked changes in [Na sup +]imeasured using a Na sup +-sensitive fluorescent probe. These changes were monitored for longer periods than in the flux studies and therefore reflect steady-state changes in [Na sup +]irather than Na sup + flux. Because halothane does not affect Na sup +, K sup +-ATPase activity in rat synaptic plasma membranes, it is unlikely that effects on this enzyme are involved in the inhibition of veratridine-evoked increases in [Na sup +]i.
Interactions between halothane and presynaptic Na sup + channels were further investigated by radioligand binding. Binding of [sup 3 H]saxitoxin to site one was not affected by halothane up to 1.2 mM. A small increase in [sup 3 H]brevetoxin-3 binding to site five was observed at moderate concentrations of halothane, whereas 1.8 mM halothane inhibited binding slightly. The significance of this biphasic effect is not known. In contrast, halothane clearly inhibited [sup 3 H]BTX-B binding to site two. Scatchard analysis was consistent with a competitive mechanism of inhibition; however, kinetic analysis revealed that 0.74 mM halothane affected both k +1 and k-1 for [sup 3 H]BTX-B binding. These findings indicate that halothane inhibits [sup 3 H]BTX-B binding by an allosteric mechanism, primarily by an increase in the dissociation rate. The effect of halothane on [sup 3 H]BTX-B binding does not appear to be due to an indirect effect on scorpion toxin binding to site three (used to enhance [sup 3 H]BTX-B binding), because halothane inhibited22Na sup + influx with similar potency in the absence or presence of scorpion venom.
Allosteric competitive inhibition of [sup 3 H]BTX-B binding also has been observed for other clinically useful drugs, including local anesthetic agents, class I anticonvulsants, class I antiarrhythmic agents, and propofol. Most of these compounds have no significant effects on neurotoxin binding to other receptor sites on the Na sup + channel. Taken together, these studies suggest a common general mechanism for Na sup + channel inhibition by several classes of drugs with distinct chemical structures mediated by a common conformational effect on the channel.
The MAC of halothane for surgical anesthesia is 0.76 vol% in humans and 1.24 vol% in rats. Corresponding aqueous halothane concentrations at 37 [degree sign] Celsius were calculated as 0.21 and 0.35 mM for humans and rats, respectively. Halothane significantly inhibited veratridine-evoked increases in22Na sup + influx, [Na sup +]i, and [sup 3 H]BTX-B binding in rat cortical synaptosomes at clinical concentrations. The potency for inhibition of [sup 3 H]BTX-B binding (IC50= 0.53 mM; 1.5 MAC) was greater than for inhibition of veratridine-evoked changes in [Na sup +]i(IC50= 0.97 mM; 2.8 MAC) and22Na sup + influx (IC50= 1.1 mM; 3.1 MAC). A similar difference was reported for phenytoin inhibition of [sup 3 H]BTX-B binding (IC50= 40 micro meter)compared with veratridine-evoked (60 micro meter)24Na sup + influx (38% inhibition at 100 micro meter)in rat brain synaptosomes. Differential Na sup + channel activation by veratridine and BTX-B probably underlies these observed differences in the inhibitory potency of halothane. The different buffers required for each assay (Na sup +-free buffer for [sup 3 H]BTX-B binding assays; 5 mM extracellular [Na sup +] for22Na sup + influx assays, and 120 mM extracellular [Na sup +] for [Na sup +]iassays) also may have contributed to the differences in halothane potency.
Small changes in Na sup + channel function can produce large shifts in equilibrium potential with profound functional consequences. The neurophysiologic significance of Na sup + channel inhibition by halothane was assessed using veratridine-evoked (Na sup + channel-dependent) release of glutamate under comparable conditions. Activation of Na sup + channels by veratridine results in sequential membrane depolarization, voltage-dependent opening of Ca2+ channels, and Ca2+-dependent exocytotic release of neurotransmitters. The rise in intracellular [Na sup +] also results in reversal of Na sup +/Ca2+ antiport and reversal of Na sup +/glutamate cotransport. Although veratridine evokes both Ca2+-independent (carrier-mediated) and -dependent (exocytotic) glutamate release from synaptosomes, the major component of release is Ca2+-dependent, similar to physiologic release; however, the pathway for Ca2+ entry is not clear and may differ from action potential-evoked Ca2+.
Inhibition of veratridine-evoked release of glutamate by halothane (IC50= 0.67 mM; 1.9 MAC) via Na sup + channel blockade could involve one or more of the following mechanisms:(1) inhibition of Na sup + channel-dependent membrane depolarization and consequently of Ca2+ channel activation;(2) inhibition of Ca2+ entry through veratridine-modified Na sup + channels ;(3) inhibition of Na sup +/glutamate transporter reversal or stimulation of glutamate reuptake; or (4) inhibition of Na sup +/Ca2+ exchange. Blockade of presynaptic Ca2+ channels coupled to release of glutamate or interference with subsequent release mechanisms also could contribute to the effect of halothane. Na sup + channel-independent mechanisms appear quantitatively less important than an effect at Na sup + channels because release of glutamate evoked by KCl, a secretogogue not dependent on Na sup + channel function, was insensitive to halothane under our assay conditions. The sensitivity of veratridine-evoked release of glutamate to halothane was comparable to that of release of glutamate evoked by 4-aminopyridine (IC50= 0.5 mM ), which also evokes Na sup + channel-dependent (tetrodotoxin-sensitive) release. Because halothane had no effect on basal or spontaneous release of glutamate, which is due primarily to reversed Na sup +/glutamate uptake, a direct effect on the Na sup +/glutamate transporter is unlikely. Stimulation of glutamate reuptake is also an unlikely mechanism because halothane (3–4 vol%) did not affect [sup 3 H]glutamate uptake into rat cortical synaptosomes significantly. [47,48]
Halothane and other volatile anesthetic agents also affect other targets, including ligand-gated ion channels, which have been proposed as principal sites for anesthetic action. For example, halothane potentiated gamma aminobutyric acid type A receptor-mediated Cl sup - current in rat hippocampal neurons (at 1.0–1.5 MAC); increased36Cl sup - uptake through gamma aminobutyric acid-gated Cl- channels (50% effective concentration [EC50]= 2.2 mM); and inhibited t-[sup 35 S]butylbicyclophosphorothionate binding to cortical membranes (IC50= 1.68 mM). These actions of halothane occur at concentrations comparable to or higher than those reported here for inhibition of Na sup + channels. Thus, Na sup + channel inhibition must also be considered as a potential target for the effects of halothane on the CNS.
Volatile anesthetic agents suppress CNS Na sup + channels in a voltage-dependent manner because of preferential interaction with inactivated channels. The allosteric inhibition by halothane (this study) and propofol of [sup 3 H]BTX-B binding is consistent with preferential anesthetic binding to the inactivated state of the Na sup + channel as described by the modulated receptor hypothesis. The inactivated conformation of the Na sup + channel appears to possess a hydrophobic drug binding site(s) that is distinct from, but allosterically coupled to, site two. Preferential binding to the inactivated Na sup + channel would favor drug effects on abnormally firing or depolarized cells over normally functioning cells as demonstrated for phenytoin, lidocaine, and lamotrigine. [38,52]Evidence suggests that volatile anesthetic agents have neuroprotective effects (for review, see ). Selective antagonism of Na sup + channels and Na sup + channel-dependent release of glutamate in repetitively active or ischemically depolarized neurons underlies the neuroprotective mechanism of several drugs and also may contribute to the neuroprotective properties of some general anesthetic agents. 
Halothane interacts with presynaptic Na sup + channels to inhibit veratridine-evoked Na sup + influx, [Na sup +]ichanges, and release of glutamate, actions which may contribute to its anesthetic and neuroprotective properties. Taken together with our previous studies of volatile anesthetic agents and propofol, [23,24]these findings emphasize that presynaptic Na sup + channels may be important targets for general anesthetic inhibition of excitatory neurotransmission in the CNS.