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).     

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 microm 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 ( approximately 2 minimum alveolar concentration; drug concentration for half-maximal inhibition [IC50] > 1.5 mM) [corrected] or propofol up to 40 microm 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 microm) and rat brain regions. Glutamate release was evoked by veratridine or increased KCl (from 5 to 35 mM) to assess the involvement of presynaptic ion channels as targets for drug actions [corrected]. CONCLUSIONS: Isoflurane and propofol inhibited Na+ channel-mediated glutamate release evoked by veratridine with greater potency than release evoked by increased KCl in synaptosomes prepared from three mammalian species and three rat brain regions. These findings are consistent with a greater sensitivity to anesthetics of presynaptic Na+ channels than of Ca2+ channels coupled to glutamate release. This widespread presynaptic action of general anesthetics is not mediated by potentiation of gamma-aminobutyric acid type A receptors, though additional mechanisms may be involved.     

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.     

Isoflurane increases the uptake of glutamate in synaptosomes from rat cerebral cortex

We have studied the effect of increasing concentrations of isoflurane on high- and low-affinity uptake of L-glutamate using synaptosomes from rat cerebral cortex. In the high-affinity uptake range, 0.5% isoflurane had no effect on uptake velocity, while 1.5% and 3.0% isoflurane caused an increase in mean Vmax to 131 (SEM 54) and 210 (103)% of control, respectively. There was no significant change in the K(m) value. Vmax and K(m) values for low-affinity uptake of L-glutamate were unchanged by 1.5% isoflurane. These results provide evidence for an isoflurane- induced increase in high-affinity uptake of glutamate into presynaptic terminals. This effect may contribute to a reduction of transmitter in the synaptic cleft and thereby decreased excitatory synaptic transmission.      

Glutamate uptake is not a major target site for anaesthetic agents

We have examined the effects of thiopentone, propofol and ketamine 3- 300 mumol litre-1, 3.6%, 2.4 rat MAC of isoflurane, 3.0%, 2.4 rat MAC of halothane and morphine 0.1-10 mumol litre-1 on uptake of [3H]glutamate into rat cerebrocortical and cerebellar synaptosomes. Corticol and cerebellar synaptosomes took up [3H]glutamate in a time-, concentration-, Na(+)-dependent and L-transpyrrolidine-2,4- dicarboxylate inhibitory manner. The Km and Vmax values for uptake were 8.6 mumol litre-1 and 1.7 nmol/min/mg protein and 2.2 mumol litre-1 and 0.7 nmol/min/mg protein in cortical and cerebellar preparations, respectively. At clinically relevant concentrations none of the agents tested influenced the uptake process. Our data suggest that the uptake of glutamate is not a major target site for anaesthetic or analgesic agents.      

Effects of Intravenous General Anesthetics on [(3) H]GABA Release from Rat Cortical Synaptosomes

Background: Potentiation by general anesthetics of [Greek small letter gamma]-aminobutyric acid (GABA)-mediated inhibitory transmission in the central nervous system is attributed to GABAA receptor-medicated postsynaptic effects. However, the role of presynaptic mechanisms in general anesthetic action is not well characterized, and evidence for anesthetic effects on GABA release is controversial. The effects of several intravenous general anesthetics on [(3) H]GABA release from rat cerebrocortical synaptosomes (isolated nerve terminals) were investigated.

Methods: Purified synaptosomes were preloaded with [(3) H]GABA and superfused with buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA metabolism and reuptake, respectively. Spontaneous and elevated potassium chloride depolarization-evoked [(3) H]GABA release were evaluated in the superfusate in the absence or presence of various anesthetics, extracellular Ca2+, GABA receptor agonists and antagonists, and 2,4-diaminobutyric acid.

Results: Propofol, etomidate, pentobarbital, and alphaxalone, but not ketamine, potentiated potassium chloride-evoked [(3) H]GABA release (by 1.3 to 2.9 times) in a concentration-dependent manner, with median effective concentration values of 5.4 +/- 2.8 [micro sign]M (mean +/- SEM), 10.1 +/- 2.1 [micro sign]M, 18.8 +/- 5.8 [micro sign]M, and 4.4 +/- 2.0 [micro sign]M. Propofol also increased spontaneous [(3) H]GABA release by 1.7 times (median effective concentration = 7.1 +/- 3.4 [micro sign]M). Propofol facilitation of [(3) H]GABA release was Ca2+ dependent and inhibited by bicuculline and picrotoxin, but was insensitive to pretreatment with 2,4-diaminobutyric acid, which depletes cytoplasmic GABA pools.     

Anesthetic Concentrations of Propofol Protect against Oxidative Stress in Primary Astrocyte Cultures: Comparison with Hypothermia

Background: The extracellular concentration of glutamate in the brain increases after oxidative damage. This increase may be caused, in part, by changes in glutamate transport by astrocytes. The authors hypothesized that propofol and hypothermia mitigate the effects on astrocytes of oxidative stress.

Methods: Primary cultures of rat cerebral astrocytes were subjected to oxidative stress by incubation with tert-butyl hydroperoxide for 30 min, followed by a 30-90-min washout period. The effects of prophylactic (simultaneous with tert-butyl hydroperoxide application) and delayed (administered 30 min after the oxidant) propofol or hypothermia were determined by measuring the uptake of glutamate as well as the release of preloaded d-aspartate (a nonmetabolizable analog of glutamate) and endogenous lactate dehydrogenase (a cytosolic marker).

Results: Delayed administration of an anesthetic concentration of propofol (1-3 [mu]m) prevented the inhibition of high-affinity glutamate uptake, stimulation of d-aspartate release, and increase in lactate dehydrogenase release caused by tert-butyl hydroperoxide (1 mm, 37[degrees]C). The protective effect of propofol (EC50 = 2 [mu]m) on glutamate uptake was 20-fold more potent than that of [alpha]-tocopherol (EC50 = 40 [mu]m). Prophylactic hypothermia (28 and 33[degrees]C) also protected astrocytes from tert-butyl hydroperoxide. Delayed hypothermia was not protective but did not compromise rescue by propofol.     

Effects of Volatile Anesthetics on Glutamate Transporter, Excitatory Amino Acid Transporter Type 3: The Role of Protein Kinase C

Background: Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. The authors studied the effects of volatile anesthetics on one type of glutamate transporters, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C in mediating these effects.

Methods: Excitatory amino acid transporter type 3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Using two-electrode voltage clamp, membrane currents were recorded before, during, and after application of l-glutamate. Responses were quantified by integrating the current trace and are reported as microcoulombs. Data are mean +/- SEM.

Results: l-Glutamate-induced responses were increased gradually with the increased concentrations of isoflurane, a volatile anesthetic. At 0.52 and 0.70 mm isoflurane, the inward current was significantly increased compared with control. Isoflurane (0.70 mm) significantly increased Vmax (maximum velocity) (3.6 +/- 0.4 to 5.1 +/- 0.4 [mu]C;P < 0.05) but not Km (Michoelis-Menten Constant) (55.4 +/- 17.0 vs. 61.7 +/- 13.6 [mu]m;P > 0.05) of EAAT3 for glutamate compared with control. Treatment of the oocytes with phorbol-12-myrisate-13-acetate, a protein kinase C activator, caused a significant increase in transporter current (1.7 +/- 0.2 to 2.5 +/- 0.2 [mu]C;P < 0.05). Responses in the presence of the combination of phorbol-12-myrisate-13-acetate and volatile anesthetics (isoflurane, halothane, or sevoflurane) were not greater than those when volatile anesthetic was present alone. Oocytes pretreated with any of the three protein kinase C inhibitors alone (chelerythrine, staurosporine, or calphostin C) did not affect basal transporter current. Although chelerythrine did not change the anesthetic effects on the activity of EAAT3, staurosporine or calphostin C abolished the anesthetic-induced increase of EAAT3 activity.     

Aging decreases the sensitivity of the GABA carrier to propofol and etomidate

The influence of aging on the pharmacodynamics of anaesthetic agents in the central nervous system remains poorly understood. As alpha- aminobutyric acid (GABA)-mediated neurotransmission appears to be an important target for anaesthetics in the brain, we hypothesized that aging could alter the sensitivity of the GABA carrier to anaesthetics. We have examined the effects of etomidate and propofol on the uptake of [3H]-GABA (5 min, 37 degrees C) into striatal synaptosomes of rats aged 2, 18 and 24 months. In 2-month-old rats, [3H]-GABA uptake was inhibited by nipecotic acid, a competitive inhibitor of the GABA carrier (IC50 = 3.6 SD 0.3 microM). Etomidate and propofol markedly reduced the activity of the GABA carrier, with IC50 values 58 (SD 3) and 46 (SD 3) microgramsmol litre-1, respectively. Aging increased IC50 values for these anaesthetics. Nipecotic acid was unaffected. These data suggest that aging selectively alters the action of etomidate and propofol in the mammalian CNS.      

Increase of Glutamate Uptake in Astrocytes: A Possible Mechanism of Action of Volatile Anesthetics

Background: Glutamate is the most ubiquitous excitatory neurotransmitter in the vertebrate central nervous system. Astrocytes play an important role in terminating glutamatergic neurotransmission by removing released glutamate from the synaptic cleft. The authors examined the effects of several anesthetics on the glutamate uptake activity of astrocytes.

Methods: Cultured astrocytes from hippocampi of rat embryos were incubated with solution containing [sup 3 H]glutamate, which was pre-equilibrated with 0-4% halothane at 37 degrees Celsius. The uptake activity was evaluated as the amount of radioactivity per cell of protein.

Results: When the reaction solution was equilibrated with 4% halothane, glutamate uptake increased to about 165% of the control. The effect of halothane was dose-dependent, and a significant augmentation (30-50%) of glutamate uptake was observed at a range in clinical use concentrations (1-2%). On the other hand, the uptake of gamma-aminobutyric acid, an inhibitory transmitter, was hardly affected by 1-4% halothane. The effect of halothane on glutamate uptake was also examined in neuron-rich culture, and similar augmentation was observed, although the extent was less than that in astrocyte culture. Biochemical subcellular fractions (i.e., glial plasmalemmal vesicles and synaptosomes) were also examined, however, only slight (not significant) increase was detected in the glutamate uptake activity. Other volatile anesthetics, such as enflurane, isoflurane, and sevoflurane, also enhanced glutamate uptake, whereas the intravenous anesthetics ketamine and pentobarbital showed no effect on glutamate uptake.     

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.     

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 [alpha]1[beta]2[gamma]2 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 [mu]m) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg).     

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.     

Neuroprotective Effects of Propofol in a Model of Ischemic Cortical Cell Cultures: Role of Glutamate and Its Transporters

Background: During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures.

Methods: Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively.

Results: At clinically relevant concentrations (0.05-10 [mu]m), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol.     

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.     

Isoflurane Enhances the Expression and Activity of Glutamate Transporter Type 3 in C6 Glioma Cells

Background: Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. Volatile anesthetics have been shown to affect glutamate transporter activity acutely (within minutes after the exposure). It is not known whether volatile anesthetics affect the expression of glutamate transporters.

Methods: Rat cultured C6 glioma cells that express excitatory amino acid transporter type 3 (EAAT3) were exposed to isoflurane at various concentrations (0.5-4.0%) or for different periods (1-24 h) at 37[degrees]C. EAAT3 mRNA, proteins, and activity were quantified.

Results: Isoflurane induced a time- and concentration-dependent increase in the mRNA and protein levels of EAAT3 in C6 cells. The maximal increase was induced by 2% isoflurane, and the cells incubated with 2% isoflurane for 3 and 7 h expressed the highest levels of EAAT3 mRNA and proteins, respectively. Similarly, glutamate uptake was higher in C6 cells exposed to 2% isoflurane for 7 h than in control cells. Actinomycin D and cycloheximide, inhibitors for mRNA and protein synthesis, respectively, did not affect the isoflurane-induced increase in EAAT3 mRNA and protein expression. Phorbol 12-myristate 13-acetate, a protein kinase C activator, also enhanced EAAT3 expression. The combination of 2% isoflurane and phorbol 12-myristate 13-acetate caused a higher level of EAAT3 expression than that induced by 2% isoflurane alone. Neither staurosporine, a protein kinase C inhibitor, nor wortmannin, a phosphatidylinositol 3 kinase inhibitor, inhibited the isoflurane-induced increase in EAAT3 expression.     

Anesthetic-induced burst suppression EEG activity requires glutamate-mediated excitatory synaptic transmission

Many anesthetics evoke electroencephalogram (EEG) burst suppression activity in humans and animals during anesthesia, and the mechanisms underlying this activity remain unclear. The present study used a rat neocortical brain slice EEG preparation to investigate excitatory synaptic mechanisms underlying anesthetic-induced burst suppression activity. Excitatory synaptic mechanisms associated with burst suppression activity were probed using glutamate receptor antagonists (CNQX and APV), GABA receptor antagonists, and simultaneous whole cell patch clamp and microelectrode EEG recordings. Clinically relevant concentrations of thiopental (50--70 microM), propofol (5--10 microM) or isoflurane (0.7--2.1 vol%, 0.5--1.5 rat minimum aveolar concentration (MAC), 200--700 microM) evoked delta slow wave activity and burst suppression EEG patterns similar to in vivo responses. These effects on EEG signals were blocked by glutamate receptor antagonists CNQX (8.6 microM) or APV (50 microM). Depolarizing intracellular bursts (amplitude=34.7+/-4.5 mV; half width=132+/-60 ms) always accompanied EEG bursts, and hyperpolarization increased intracellular burst amplitudes. Barrages of glutamate-mediated excitatory events initiated EEG bursting activity. Glutamate-mediated excitatory postsynaptic currents were significantly depressed by higher anesthetic concentrations that depressed burst suppression EEG activity. A GABA(A) agonist produced a similar EEG effect to the anesthetics. It appears that anesthetic effects at both glutamate and GABA synapses contribute to EEG patterns seen during anesthesia.     

The mechanisms of depression by benzodiazepines, barbiturates and propofol of excitatory synaptic transmissions mediated by adenosine neuromodulation

Adenosine is known to modulate synaptic functions through adenosine receptors and the related adenosine neuromodulatory system. Benzodiazepines, barbiturates and propofol, the main actins of which are facilitation of GABAA receptor functions, also facilitate accumulation of endogenous adenosine in the extracellular space by inhibiting adenosine uptake via adenosine transporters. The accumulated adenosine depresses excitatory synaptic transmission by decreasing transmitter release, depressing postsynaptic sensitivity and inhibiting neuronal excitability through adenosine A1 receptors and the related adenosine neuromodulatory system. The adenosine-induced depressions of excitatory synaptic transmissions probably contribute to the mechanisms of the anesthetic, sedative, anticonvulsant and neuroprotective effects of these anesthetics and sedatives. An understanding of the synaptic mechanisms of these anesthetics and sedatives through the adenosine neuromodulatory system is needed for rational clinical use of the drugs.     

Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters

BACKGROUND: During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures. METHODS: Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively. RESULTS: At clinically relevant concentrations (0.05-10 microm), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol. CONCLUSION: Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.     

Excitatory Synaptic Transmission Mediated by NMDA Receptors Is More Sensitive to Isoflurane than Are Non-NMDA Receptor-mediated Responses

Background: Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor-mediated synaptic responses in slices from the rat hippocampus.

Methods: Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor-mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents.

Results: Clinically relevant concentrations of halothane (1.2 vol% [almost equal to] 0.35 mM) depressed fEPSP amplitudes mediated by NMDA receptors and non-NMDA receptors to a similar degree (mean +/- SD: 63.3 +/- 14.0% of control, n = 5; 60.2 +/- 7.3% of control, n = 7, respectively). In contrast, isoflurane (1.4 vol% [almost equal to] 0.50 mM) preferentially depressed fEPSP amplitudes mediated by NMDA receptors (44.0 +/- 7.4% of control, n = 6, P < 0.001) compared with those for non-NMDA receptors (68.7 +/- 5.4% of control, n = 6), indicating a selective, additional postsynaptic effect. Paired pulse facilitation of fEPSPs was increased significantly by both anesthetic agents from 1.37 +/- 0.13 to 1.91 +/- 0.25 (n = 5, P < 0.05 for halothane) and from 1.44 +/- 0.04 to 1.64 +/- 0.08 (n = 5, P < 0.01 for isoflurane), suggesting that presynaptic mechanisms are also involved in fEPSP depression produced by the anesthetic agents. Neither rise times nor decay times of fEPSPs were changed in the presence of the anesthetic agents.