Widespread Inhibition of Sodium Channel-dependent Glutamate Release from Isolated Nerve Terminals by Isoflurane and Propofol

Background : Controversy persists concerning the mechanisms and role of general anesthetic inhibition of glutamate release from nerve endings. To determine the generality of this effect and to control for methodologic differences between previous studies, the authors analyzed the presynaptic effects of isoflurane and propofol on glutamate release from nerve terminals isolated from several species and brain regions.

Methods : Synaptosomes were prepared from rat, mouse, or guinea pig cerebral cortex and also from rat striatum and hippocampus. Release of endogenous glutamate evoked by depolarization with 20 [mu]m veratridine (which opens voltage-dependent Na+ channels by preventing inactivation) or by 30 mm KCl (which activates voltage-gated Ca2+ channels by membrane depolarization) was monitored using an on-line enzyme-linked fluorometric assay.

Results : Glutamate release evoked by depolarization with increased extracellular KCl was not significantly inhibited by isoflurane up to 0.7 mm (~2 minimum alveolar concentration; drug concentration for half-maximal inhibition > 1.5 mm) or propofol up to 40 [mu]m in synaptosomes prepared from rat, mouse, or guinea pig cerebral cortex, rat hippocampus, or rat striatum. Lower concentrations of isoflurane or propofol significantly inhibited veratridine-evoked glutamate release in all three species (isoflurane IC50 = 0.41-0.50 mm; propofol IC50 = 11-18 [mu]m) and rat brain regions. Inhibition of veratridine-evoked release was insensitive to the [gamma]-aminobutyric acid receptor type A antagonist bicuculline (100 [mu]m) in rat cortical synaptosomes.     

Effects of Propofol on Sodium Channel-dependent Sodium Influx and Glutamate Release in Rat Cerebrocortical Synaptosomes

Background: Previous electrophysiologic studies have implicated voltage-dependent Na+ channels as a molecular site of action for propofol. This study considered the effects of propofol on Na+ channel-mediated Na+ influx and neurotransmitter release in rat brain synaptosomes (isolated presynaptic nerve terminals).

Methods: Purified cerebrocortical synaptosomes from adult rats were used to determine the effects of propofol on Na+ influx through voltage-dependent Na+ channels (measured using22 Na+) and intracellular [Na+] (measured by ion-specific spectrofluorimetry). For comparison, the effects of propofol on synaptosomal glutamate release evoked by 4-aminopyridine (Na+ channel dependent), veratridine (Na (+) channel dependent), and KCl (Na+ channel independent) were studied using enzyme-coupled fluorimetry.

Results: Propofol inhibited veratridine-evoked22 Na+ influx (inhibitory concentration of 50% [IC50] = 46 micro Meter; 8.9 micro Meter free) and changes in intracellular [Na+] (IC50 = 13 micro Meter; 6.3 micro Meter free) in synaptosomes in a dose-dependent manner. Propofol also inhibited 4-aminopyridine-evoked (IC50 = 39 micro Meter; 19 micro Meter free) and veratridine (20 micro Meter)-evoked (IC (50) = 30 micro Meter; 14 micro Meter free), but not KCl-evoked (up to 100 micro Meter) glutamate release from synaptosomes.     

General anesthetics do not affect release of the neuropeptide cholecystokinin from isolated rat cortical nerve terminals

BACKGROUND: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized. METHODS: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35 degrees C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration. RESULTS: Elevation of extracellular KCl concentration (to 15-30 mM) or veratridine (10-20 microm) stimulated Ca2+ -dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 microm), pentobarbital (50 and 100 microm), thiopental (20 microm), etomidate (20 microm), ketamine (20 microm), isoflurane (0.6-0.8 mM), or halothane (0.6-0.8 mMm). CONCLUSIONS: Clinically relevant concentrations of several classes of general anesthetics did not affect basal, KCl-evoked, or veratridine-evoked CCK8s release from isolated rat cortical nerve terminals. This is in contrast to the demonstrable effects of certain general anesthetics on the release of amino acid and catecholamine transmitters. These transmitter-specific presynaptic effects of general anesthetics suggest that anesthetic-sensitive presynaptic targets are not common to all transmitter classes.     

Differential effects of anesthetic and nonanesthetic cyclobutanes on neuronal voltage-gated sodium channels

BACKGROUND: Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods. METHODS: Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording. RESULTS: F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [approximately 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [approximately 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50 = 0.5 mM [approximately 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70+/-9% block at 0.6 mM [approximately 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21+/-9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16+/-2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation. CONCLUSIONS: The anesthetic cyclobutane F3 significantly inhibited Na+ channel-mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.     

Inhibition of Presynaptic Sodium Channels by Halothane

Background: Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na sup +) channels. In this study, the biochemical effects of halothane on Na sup + channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na sup + channels in anesthetic effects.

Methods: Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na sup + channel-dependent Na sup + influx (using22 Na sup +), changes in intrasynaptosomal [Na sup +] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na sup + 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 sup + channel-dependent glutamate release (using enzyme-coupled spectrofluorometry).

Results: Halothane inhibited veratridine-evoked22 Na sup + influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na sup +] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [sup 3 H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na sup + 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).     

Effects of isoflurane and propofol on glutamate and GABA transporters in isolated cortical nerve terminals

BACKGROUND: Depression of glutamate-mediated excitatory transmission and potentiation of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission appear to be primary mechanisms by which general anesthetics produce anesthesia. Since effects on transmitter transport have been implicated in anesthetic actions, the authors examined the sensitivity of presynaptic glutamate and GABA transporters to the effects of a representative volatile (isoflurane) and a representative intravenous (propofol) anesthetic. METHODS: A dual-isotope (l-[3H]glutamate and [14C]GABA) approach allowed simultaneous comparisons of anesthetic effects on three independent assays of glutamate and GABA transporters in adult rat cerebral cortex: transmitter uptake into isolated nerve terminals (synaptosomes), transmitter binding to lysed and washed synaptosomes (synaptic membranes), and carrier-mediated release (reverse transport) of transmitter from preloaded synaptosomes using a modified superfusion system. RESULTS: Isoflurane produced small but statistically significant inhibition of l-[3H]glutamate and [14C]GABA uptake, while propofol had no effect. Inhibition of uptake by isoflurane was noncompetitive, an outcome that was mimicked by indirectly affecting transporter function through synaptosomal depolarization. Neither isoflurane nor propofol affected l-[3H]glutamate or [14C]GABA binding to synaptic membranes or Ca(2+)-independent carrier-mediated l-[3H]glutamate or [14C]GABA release (reverse transport). CONCLUSIONS: These findings suggest that isoflurane and propofol at clinical concentrations do not affect excitatory glutamatergic transmission or inhibitory GABAergic transmission directly effects on their presynaptic neuronal transporters.     

Inhibition by Volatile Anesthetics of Endogenous Glutamate Release from Synaptosomes by a Presynaptic Mechanism

Background: Synaptic transmission is more sensitive than axonal conduction to the effects of general anesthetics. Previous studies of the synaptic effects of general anesthetics have focused on postsynaptic sites of action. We now provide direct biochemical evidence for a presynaptic effect of volatile anesthetics on neurotransmitter release.

Methods: Rat cerebrocortical synaptosomes (isolated presynaptic nerve terminals) were used to determine the effects of general anesthetics on the release of endogenous L-glutamate, the major fast excitatory neurotransmitter. Basal and evoked (by 4-aminopyridine, veratridine, increased KCl, or ionomycin) glutamate release were measured by continuous enzyme-coupled fluorometry.

Results: Clinical concentrations of volatile halogenated anesthetics, but not of pentobarbital, inhibited 4-aminopyridine-evoked Calcium2+ -dependent glutamate release. Halothane also inhibited veratridine-evoked glutamate release but not basal, KCl-evoked, or ionomycin-evoked glutamate release. Halothane inhibited both the 4-aminopyridine-evoked and the KCl-evoked increase in free intrasynaptosomal [Calcium2+].     

Effects of Isoflurane and Propofol on Glutamate and GABA Transporters in Isolated Cortical Nerve Terminals

Background: Depression of glutamate-mediated excitatory transmission and potentiation of [gamma]-aminobutyric acid (GABA)-mediated inhibitory transmission appear to be primary mechanisms by which general anesthetics produce anesthesia. Since effects on transmitter transport have been implicated in anesthetic actions, the authors examined the sensitivity of presynaptic glutamate and GABA transporters to the effects of a representative volatile (isoflurane) and a representative intravenous (propofol) anesthetic.

Methods: A dual-isotope (l-[3H]glutamate and [14C]GABA) approach allowed simultaneous comparisons of anesthetic effects on three independent assays of glutamate and GABA transporters in adult rat cerebral cortex: transmitter uptake into isolated nerve terminals (synaptosomes), transmitter binding to lysed and washed synaptosomes (synaptic membranes), and carrier-mediated release (reverse transport) of transmitter from preloaded synaptosomes using a modified superfusion system.

Results: Isoflurane produced small but statistically significant inhibition of l-[3H]glutamate and [14C]GABA uptake, while propofol had no effect. Inhibition of uptake by isoflurane was noncompetitive, an outcome that was mimicked by indirectly affecting transporter function through synaptosomal depolarization. Neither isoflurane nor propofol affected l-[3H]glutamate or [14C]GABA binding to synaptic membranes or Ca2+-independent carrier-mediated l-[3H]glutamate or [14C]GABA release (reverse transport).     

Differential Effects of Anesthetic and Nonanesthetic Cyclobutanes on Neuronal Voltage-gated Sodium Channels

Background: Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods.

Methods: Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording.

Results: F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [~ 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [~ 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50= 0.5 mM [~ 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70 +/- 9% block at 0.6 mM [~ 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21 +/- 9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16 +/- 2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation.     

General Anesthetics Do Not Affect Release of the Neuropeptide Cholecystokinin from Isolated Rat Cortical Nerve Terminals

Background: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized.

Methods: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35[degrees]C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration.

Results: Elevation of extracellular KCl concentration (to 15-30 mm) or veratridine (10-20 [mu]m) stimulated Ca2+-dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 [mu]m), pentobarbital (50 and 100 [mu]m), thiopental (20 [mu]m), etomidate (20 [mu]m), ketamine (20 [mu]m), isoflurane (0.6-0.8 mm), or halothane (0.6-0.8 mm).     

The effects of general anesthetics on norepinephrine release from isolated rat cortical nerve terminals

Intravenous and volatile general anesthetics inhibit norepinephrine (NE) release from sympathetic neurons and other neurosecretory cells. However, the actions of general anesthetics on NE release from central nervous system (CNS) neurons are unclear. We investigated the effects of representative IV and volatile anesthetics on [(3)H]NE release from isolated rat cortical nerve terminals (synaptosomes). Purified synaptosomes prepared from rat cerebral cortex were preloaded with [(3)H]NE and superfused with buffer containing pargyline (a monoamine oxidase inhibitor) and ascorbic acid (an antioxidant). Basal (spontaneous) and stimulus-evoked [(3)H]NE release was evaluated in the superfusate in the absence or presence of various anesthetics. Depolarization with increased concentrations of KCl (15-20 mM) or 4-aminopyridine (0.5-1.0 mM) evoked concentration- and Ca(2+)-dependent increases in [(3)H]NE release from rat cortical synaptosomes. The IV anesthetics etomidate (5-40 microM), ketamine (5-30 microM), or pentobarbital (25-100 microM) did not affect basal or stimulus-evoked [(3)H]NE release. Propofol (5-40 microM) increased basal [(3)H]NE release and, at larger concentrations, reduced stimulus-evoked release. The volatile anesthetic halothane (0.15-0.70 mM) increased basal [(3)H]NE release, but did not affect stimulus-evoked release. These findings demonstrate drug-specific stimulation of basal NE release. Noradrenergic transmission may represent a presynaptic target for selected general anesthetics in the CNS. Given the contrasting effects of general anesthetics on the release of other CNS transmitters, the presynaptic actions of general anesthetics are both drug- and transmitter-specific. IMPLICATIONS: General anesthetics affect synaptic transmission both by altering neurotransmitter release and by modulating postsynaptic responses to transmitter. Anesthetics exert both drug-specific and transmitter-specific effects on transmitter release: therapeutic concentrations of some anesthetics stimulate basal, but not evoked, norepinephrine release, in contrast to evoked glutamate release, which is inhibited.     

Anesthetics Affect the Uptake but Not the Depolarization-evoked Release of GABA in Rat Striatal Synaptosomes

Background: Numerous classes of anesthetic agents have been shown to enhance the effects mediated by the postsynaptic gamma-aminobutyric acid A (GABAA) receptor-coupled chloride channel in the mammalian central nervous system. However, presynaptic actions of anesthetics potentially relevant to clinical anesthesia remain to be clarified. Therefore, in this study, the effects of intravenous and volatile anesthetics on both the uptake and the depolarization-evoked release of GABA in the rat stratum were investigated.

Methods: Assay for specific GABA uptake was performed by measuring the radioactivity incorporated in purified striatal synaptosomes incubated with3 H-GABA (20 nM, 5 min, 37 degrees Celsius) and increasing concentrations of anesthetics in either the presence or the absence of nipecotic acid (1 mM, a specific GABA uptake inhibitor). Assay for GABA release consisted of superfusing3 H-GABA preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml *symbol* min sup 1, 37 degrees Celsius) and measuring the radioactivity obtained from 0.5 ml fractions over 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of either KCl alone (9 mM, 15 mM) or with various concentrations of anesthetics (5 min), and finally, with no pharmacologic stimulation (5 min). The following anesthetic agents were tested: propofol, etomidate, thiopental, ketamine, halothane, enflurane, isoflurane, and clonidine.

Results: More than 95% of3 H-GABA uptake was blocked by a 10 sup 3 -M concentration of nipecotic acid. Propofol, etomidate, thiopental, and ketamine induced a dose-related, reversible, noncompetitive, inhibition of3 H-GABA uptake: IC50 = 4.6 plus/minus 0.3 x 105 M, 5.8 plus/minus 0.3 x 10 sup -5 M, 2.1 plus/minus 0.4 x 10 sup -3 M, and 4.9 plus/minus 0.5 x 10 sup -4 M for propofol, etomidate, thiopental, and ketamine, respectively. Volatile agents and clonidine had no significant effect, even when used at concentrations greater than those used clinically. KCl application induced a significant, calcium-dependent, concentration-related, increase from basal3 H-GABA release, +34 + 10% (P < 0.01) and +61 plus/minus 13% (P < 0.001), respectively, for 9 mM and 15 mM KCl. The release of3 H-GABA elicited by KCl was not affected by any of the anesthetic agents tested.     

Halothane and isoflurane increase spontaneous but reduce the N-methyl-D-aspartate-evoked dopamine release in rat striatal slices: evidence for direct presynaptic effects

BACKGROUND: Experimental data suggest that volatile anesthetics induce significant changes in extracellular dopamine concentrations in the striatum, a restricted but functionally important brain area. In the present study, the authors used a superfused slice preparation to examine the effects of halothane and isoflurane on both spontaneous and N-methyl-D-aspartate (NMDA)-evoked dopamine release in the striatum, and whether these effects involved actions of these anesthetics mediated by gamma-aminobutyric acid receptors in this structure. METHODS: Radioactivity collected from 5-min fractions was compared in the absence (basal release) or presence (evoked release) of NMDA alone and combined with various pharmacologic or anesthetic agents in slices of the dorsolateral striatum and synaptosomes of the whole striatum preloaded with 3H-dopamine and superfused with artificial cerebrospinal fluid. RESULTS: In tetrodotoxin-treated striatal slices, halothane and isoflurane significantly increased dopamine basal release (EC50 = 0.33 mM and 0.41 mM for halothane and isoflurane, respectively). Both agents decreased the NMDA-evoked dopamine release in both the absence (IC50 = 0.15 mM and 0.14 mM for halothane and isoflurane, respectively) and presence (IC50 = 0.15 mM for both halothane and isoflurane) of tetrodotoxin in slices, and in synaptosomes (IC50 = 0.19 mM for both halothane and isoflurane). NMDA-induced dopamine release was significantly enhanced by bicuculline, a gamma-aminobutyric acid receptor antagonist. Halothane and isoflurane inhibitory effects on NMDA-evoked dopamine release were significantly reduced in the presence of bicuculline. CONCLUSION: These results indicate that halothane and isoflurane decrease the NMDA-evoked dopamine release by acting directly at dopamine terminals in striatal slices. They support the involvement of both depression of presynaptic NMDA receptor-mediated responses and enhancement of gamma-aminobutyric acid receptor-mediated responses in these effects.     

Halothane and Isoflurane Increase Spontaneous but Reduce the N-methyl-D-aspartate-evoked Dopamine Release in Rat Striatal Slices: Evidence for Direct Presynaptic Effects

Background: Experimental data suggest that volatile anesthetics induce significant changes in extracellular dopamine concentrations in the striatum, a restricted but functionally important brain area. In the present study, the authors used a superfused slice preparation to examine the effects of halothane and isoflurane on both spontaneous and N-methyl-D-aspartate (NMDA)-evoked dopamine release in the striatum, and whether these effects involved actions of these anesthetics mediated by [gamma]-aminobutyric acid receptors in this structure.

Methods: Radioactivity collected from 5-min fractions was compared in the absence (basal release) or presence (evoked release) of NMDA alone and combined with various pharmacologic or anesthetic agents in slices of the dorsolateral striatum and synaptosomes of the whole striatum preloaded with 3H-dopamine and superfused with artificial cerebrospinal fluid.

Results: In tetrodotoxin-treated striatal slices, halothane and isoflurane significantly increased dopamine basal release (EC50 = 0.33 mM and 0.41 mM for halothane and isoflurane, respectively). Both agents decreased the NMDA-evoked dopamine release in both the absence (IC50 = 0.15 mM and 0.14 mM for halothane and isoflurane, respectively) and presence (IC50 = 0.15 mM for both halothane and isoflurane) of tetrodotoxin in slices, and in synaptosomes (IC50 = 0.19 mM for both halothane and isoflurane). NMDA-induced dopamine release was significantly enhanced by bicuculline, a [gamma]-aminobutyric acid receptor antagonist. Halothane and isoflurane inhibitory effects on NMDA-evoked dopamine release were significantly reduced in the presence of bicuculline.     

Different effects of volatile anesthetics and polyhalogenated alkanes on depolarization-evoked glutamate release in rat cortical brain slices.

Anesthetics cause a reduction in excitatory neurotransmission that may be important in the mechanisms of in vivo anesthetic action. Because glutamate is the major excitatory neurotransmitter in mammalian brain, evaluation of anesthetic effects on induced glutamate release is relevant for studying this potential mechanism of anesthetic action. In the present study, we compared the effects of anesthetics and nonanesthetics (halogenated alkanes that disobey the Meyer-Overton hypothesis) on depolarization-evoked glutamate release. Glutamate released from rat cortical brain slices after chemically induced depolarization (50 mM KCl) was measured continuously using an enzymatic fluorescence assay. The effects of the volatile anesthetics isoflurane and enflurane were compared with the effects of the transitional compound 1,1,2-trichlorotrifluoroethane, the nonanesthetic compound 1,2-dichlorohexafluorocyclobutane, and other polyhalogenated alkanes. Tested concentrations included effective anesthetic concentrations for the anesthetics and transitional compounds, and concentrations predicted to be anesthetic based on lipid solubility for the nonanesthetics. Isoflurane dose-dependently reduced depolarization-evoked glutamate release in cortical brain slices. Isoflurane and enflurane at concentrations equivalent to 1 minimum alveolar anesthetic concentration (MAC) reduced the KCl-evoked release to 20% and 17% of control, respectively. The transitional compound 1,1,2-trichlorotrifluoroethane at 210 microM (approximately 1.2 MAC) reduced glutamate release to 47%, and the nonanesthetic 1,2-dichlorohexafluorocyclobutane increased glutamate release at 70 microM (approximately 3 MAC). These findings support the hypothesis that the modulation of excitatory neurotransmission might be responsible, in part, for in vivo anesthetic action. IMPLICATIONS: The volatile anesthetics isoflurane and enflurane reduce depolarization-evoked glutamate release in rat brain slices. The transitional compound 1,1,2-trichlorotrifluoroethane reduces glutamate release to a much lesser extent, and the nonanesthetic 1,2-dichlorohexafluorocyclobutane does not reduce glutamate release. These findings support the hypothesis that the modulation of excitatory neurotransmission might be responsible, in part, for in vivo anesthetic action.     

Halothane and Isoflurane Differentially Affect the Regulation of Dopamine and Gamma-aminobutyric Acid Release Mediated by Presynaptic Acetylcholine Receptors in the Rat Striatum

Background: General anesthetics are thought to produce their hypnotic effects mainly by acting at ligand-gated ionic channels in the central nervous system (CNS). Although it is well established that volatile anesthetics significantly modify the activity of the acetylcholine nicotinic receptors of the neuromuscular junction, little is known about their actions on the acetylcholine receptors in the CNS. In this study, the effects of halothane and isoflurane on the regulation of dopamine (DA) (gamma-aminobutyric acid [GABA]) depolarization-evoked release mediated by nicotinic (muscarinic) presynaptic receptors were studied in the rat striatum.

Methods: Assay for GABA (dopamine) release consisted of3 H-GABA (sup 3 H-DA)-preloaded synaptosomes with artificial cerebrospinal fluid (0.5 ml/min, 37 degrees Celsius) and measuring the radioactivity obtained from 1-min fractions for 18 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of depolarizing agents combined with vaporized halothane and isoflurane (0.5-5%, 5 min), and finally with no pharmacologic stimulation (5 min). The depolarizing agents were potassium chloride (KCl; 9 mM) alone or with acetylcholine (10 sup -6 - 10 sup -4 M) and/or atropine (10 sup -5 M) for experiments with3 H-GABA, and KCl (15 mM) and nicotine (10 sup -7 - 5 x 10 sup -4 M) alone or with mecamylamine (10 sup -5 M) for experiments with3 H-DA.

Results: Potassium chloride induced a significant, Ca2+ -dependent release of both3 H-GABA and3 H-DA. Nicotine produced a concentration-related, mecamylamine-sensitive3 H-DA release that was significantly attenuated by nicotine (10 sup -7 M) preincubation. Acetylcholine elicited a dose-dependent, atropine-sensitive reduction of the KCl-evoked3 H-GABA release. Halothane and isoflurane significantly decreased the nicotine-evoked3 H-DA release but had only limited depressant effects on the KCl-stimulated3 H-DA and no action on the KCl-induced3 H-GABA release. The effects of acetylcholine on3 H-GABA release were reversed by halothane but not by isoflurane.     

The effect of isoflurane and sevoflurane on cerebrocortical presynaptic Ca2+ and protein kinase C activity

Protein kinase C (PKC) is an important enzyme involved in the regulation of neurotransmission and might also be important in the mediation of ischemic neuronal death. PKC has been implicated as a target of volatile anesthetics as well as in anesthetic protection against ischemia. The present study tested the effect of isoflurane and sevoflurane, both used in neuroanesthetic practice, on presynaptic free cytosolic Ca2+ ([Ca2+](i)) and PKC activity. To measure [Ca2+](i) and PKC activation simultaneously, rat synaptosomes, mostly containing presynaptic terminals, were loaded with the fluorescent probes fura-2 and fim-1, respectively. The synaptosomes were exposed to either isoflurane or sevoflurane in concentrations corresponding to 1 and 2 MAC values in rats, both in Ca2+-containing and Ca2+-free medium. After 8 minutes of anesthetic exposure, 1 mM 4-aminopyridine was added to induce membrane depolarization. Isoflurane 1 and 2 MAC increased the basal PKC activity after 8 minutes in Ca2+-containing medium by 15.1% (3.6%) and 30.5% (5.5%) compared with control, respectively [mean (SEM); n = 9, both values P < 0.05]. Sevoflurane 2 MAC transiently decreased but thereafter increased the PKC activity (P < 0.05). In Ca2+ -free medium sevoflurane attenuated the PKC activity (P < 0.05). The anesthetics did not alter the depolarization-evoked enzyme activation. Furthermore, 2 MAC of both isoflurane and sevoflurane increased the basal- and attenuated the depolarization-evoked increase in [Ca2+](i) (P < 0.05). The present study supports the hypotheses that volatile anesthetics affect presynaptic PKC activity and that the anesthetic effect on enzyme activation seems to be related to an increase in [Ca2+](i).     

Riluzole Blocks Dopamine Release Evoked by N-methyl-D-aspartate, Kainate, and Veratridine in the Rat Striatum

Background: Dopamine (DA) is released in large amounts during cerebral ischemia and may exacerbate tissue damage. Riluzole (54274 RP) is a recently developed agent that depresses glutamate neurotransmission in the central nervous system (CNS) and that may protect against ischemic injury in some animal models. Because glutamate stimulates the release of DA in the striatum, the authors hypothesized that riluzole could antagonize DA release in this structure.

Methods: Assay for DA release consisted of superfusing3 H-DA preloaded synaptosomes with artificial cerebrospinal fluid (1 ml/min, 37 [degree sign] Celsius) and measuring the radioactivity obtained from 1-min fractions over 22 min, first in the absence of any treatment (spontaneous release, 8 min), then in the presence of depolarizing agents combined with riluzole (0.1-100 micro Meter, 5 min), and finally with no pharmacologic stimulation (9 min). The following depolarizing agents were tested: KCl (9, 15 mM), veratridine (0.01-1 micro Meter), N-methyl-D-aspartate (NMDA, 0.1-1 mM), kainate (0.1-1 mM), and nicotine (0.01-0.5 mM). Assay for DA uptake was performed by measuring the radioactivity incorporated in synaptosomes incubated with3 H-DA (44 nM; 5 min; 37 [degree sign] Celsius).

Results: All depolarizing agents produced a significant, concentration-related increase from basal3 H-DA release. Riluzole was found to decrease the release induced by veratridine (1 micro Meter), NMDA (1 mM), and kainate (1 mM) in a significant, concentration-related manner (IC50 = 9.5 micro Meter, 1.6 micro Meter, and 5.8 micro Meter for veratridine, NMDA, and kainate, respectively). In contrast, it did not affect the release elicited by either KCl or nicotine. Riluzole had no significant effect on the specific3 H-DA uptake.     

Anesthetic properties of 4-iodopropofol: implications for mechanisms of anesthesia

BACKGROUND: Positive modulation of gamma-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative-anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor-dependent and -independent anesthetic actions. METHODS: The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA alpha1beta2gamma2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. RESULTS: Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel-mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 +/- 0.5 microM) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg). CONCLUSIONS: Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.     

Isoflurane decreases ATP sensitivity of guinea pig cardiac sarcolemmal KATP channel at reduced intracellular pH

BACKGROUND: Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. METHODS: The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. RESULTS: At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. CONCLUSIONS: Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.