Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Schwartz, Rochelle D.; Jackson, John L.; Weigert, D; Skolnick, Phil; Paul, Soumen

doi:10.1523/jneurosci.05-11-02963.1985

Cited by 94 publications

(45 citation statements)

References 33 publications

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“…Pharmacologically relevant concentrations of intravenous anesthetics (e.g., barbiturates, etomidate, alphaxalone, propofol) produce multiple actions at GABAA receptors (Ashton et al, 1981;Schwartz et al, 1985;Moody et al, 1988;Lin et al, 1992). Although previous studies have demonstrated that a /3 subunit is required for the GABA-positive actions of a range of structurally unrelated intravenous agents in recombinant receptors composed of a 1/32/3 y2 subunits (Harris et a!., 1995), this report identifies a specific amino acid necessary for action of etomidate at this receptor.…”

Section: Discussionmentioning

confidence: 99%

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors

Moody

Knauer

Granja

et al. 1997

Journal of Neurochemistry

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Most general anesthetics produce two distinct actions at GABAA receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a /3 subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABAA receptors, site-directed mutagenesis of the /33 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABAA receptor composed of al/33y2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate { 3H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [3H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on /33(Asn290) indicates that the dual actions of this intravenous anesthetic at GABAA receptors are mediated via distinct loci. Key Words: Etomidate-Anesthetics-GABAA receptors-Chloride currents-Flunitrazepam. J. Neurochem. 69, 1310Neurochem. 69, -1313Neurochem. 69, (1997.Although the molecular targets responsible for anesthesia remain controversial (Franks and Lieb, 1994), the ability of intravenous anesthetics to affect GABAA receptor function has been recognized for over 15 years (Skolnick et al., 1980;Study and Barker, 1981;Leeb-Lundberg and Olsen, 1982). Both electrophysiological and neurochemical studies have shown that, within a clinically relevant concentration range, intravenous anesthetics such as pentobarbital, propofol, and etomidate produce two characteristic actions at GABAA receptors (Skolnick et al., 1981;Study and Barker, 1981;Sanna et al., 1995; Hill-Venning et al., 1997). Typically, at concentrations that have little or no effect on chloride currents, these agents augment GABA-gated currents. This effect, often referred to as an "indirect action," is also characteristic of other drugs with sedative/hypnotic properties [e.g., diazepam (Sigel et al., 1990) and phenobarbital (Schulz andMacDonald, 1981)1 that are by themselves not efficacious anesthetic agents. At higher concentrations, intravenous anesthetics have a direct action, increasing chloride conductance in the nominal absence of GABA (Study and Barker, 1981;Sigel et al., 1990), suggesting that this direct effect may be related to the process of anesthesia.There are often marked differences among brain regions in the action of anesthetics at GABAA receptors (Ashton et al., 1981;Harris et al., 1993), and such differences are likely to reflect the regional heterogeneity of GABAA receptors (Wisden et al., 1992). This hypothesis is consistent with studies in recombinant GABAA receptors demonstrating that subunit composition has a dramatic effect on drug action (Sigel et al., 1990;Luddens et al., 1994). For example, although both volatile and ...

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Section: Discussionmentioning

confidence: 99%

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors

Moody

Knauer

Granja

et al. 1997

Journal of Neurochemistry

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“…Radioactivity was determined by liquid scintillation spectrometry. A full characterization of the effects of various barbiturates and GABA receptor agonists and antagonists on 36CP-efflux and uptake has been reported elsewhere (29)(30)(31)33 (34) and of Rohde and Harris (35). RESULTF Addition of ethanol to cerebral cortical synaptoneurosomes stimulated 36CJ-uptake in a dose-dependent fashion (Fig.…”

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confidence: 89%

“…Recently, we have reported the use of the "synaptoneurosome," a subcellular brain preparation, to measure GABA/ barbiturate receptor-mediated Cl-transport in vitro (29)(30)(31). Morphologic characterization of this preparation has revealed the presence ofboth pre-and postsynaptic membranes that form closed vesicles (32 (29,30) for measurement of 36C-efflux.…”

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confidence: 99%

“…Morphologic characterization of this preparation has revealed the presence ofboth pre-and postsynaptic membranes that form closed vesicles (32 (29,30) for measurement of 36C-efflux. Cerebral cortices from adult male Sprague-Dawley rats (200-250 g) housed under diurnal lighting conditions with free access to food and water were removed, pooled, and dissected free from surrounding white matter, Brain tissue (1 g) was homogenized in 7 vol (wt/vol) of an ice-cold buffer containing 20 mM Hepes/Tris, 118 mM NaCl, 4.7 mM KCl, 1.18 mM MgSO4, and 2.5 mM CaCl2 (pH 7.4) using a glass-glass homogenizer (five strokes).…”

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confidence: 99%

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Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes.

Suzdak¹,

Schwartz²,

Skolnick³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The effects of ethanol on Cl-uptake were studied using a cell-free subcellular preparation from brain that contains a y-aminobutyric acid (GABA)/barbiturate receptor-sensitive Cl transport system. In isolated vesicles prepared from rat cerebral cortex, ethanol, at concentrations that are present during acute intoxication (20-50 mM), stimulated 36CI-uptake in a concentration-dependent and biphasic manner. The ethanol-stimulated uptake of 36CI-was markedly inhibited by the GABA antagonists picrotoxin and bicuculline but not by a variety of other neurotransmitter receptor antagonists. The effects of ethanol in stimulating 36CI-uptake in isolated brain vesicles were qualitatively and quantitatively similar to that of pentobarbital. Ethanol also markedly potentiated both muscimol-and pentobarbital-stimulated 36CI-uptake at concentrations below those that directly stimulate Cl-uptake. Under our incubation conditions, ethanol did not release GABA, suggesting that it interacts with the postsynaptic GABA/barbiturate receptor complex. The ability of ethanol to stimulate GABA/barbiturate receptor-mediated Cl-transport may explain many of its pharmacological properties and provides a mechanism for the common psychopharmacological actions of ethanol, barbiturates, and benzodiazepines.Ethanol is one of the oldest and most commonly used of all psychotropic drugs (1). Repeated exposure to ethanol produces both psychological and physical dependence and its abuse potential constitutes a major public health problem (2). The neurochemical mechanism(s) underlying the depressant effects of ethanol on the central nervous system (CNS) is poorly understood (3) despite numerous studies demonstrating effects of ethanol on several neurotransmitter systems (4, 5). Ethanol shares many pharmacologic actions with both barbiturates and benzodiazepines. For example, ethanol, like barbiturates and benzodiazepines, possesses anxiolytic and sedative/hypnotic activity in both laboratory animals (6, 7) and humans (8). Moreover, previous studies have documented the development of behavioral cross-tolerance between ethanol, barbiturates, and benzodiazepines (9, 10). Benzodiazepines and barbiturates, which also show crossdependence with each other, are effective in alleviating the withdrawal symptoms that occur after chronic ethanol administration, suggesting that all three drugs may share a common mechanism of action (11).It is now generally accepted that both benzodiazepines and barbiturates produce their major pharmacological effects by augmenting the actions of the principal inhibitory neurotransmitter of brain, y-aminobutyric acid (GABA) (12)(13)(14)(15) ly associated with the Cl-channel in both rat and mouse brain membranes (25,26). Unfortunately, the concentrations of ethanol used in many of these receptor binding studies exceed those observed during acute intoxication (>30 mM) and are, in fact, many times above the lethal concentration (>100 mM) (27,28).Recently, we have reported the use of the "synaptoneurosome," a subcellular brain pre...

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“…Biochemical studies suggest that barbiturates exert these effects at specific recognition sites located at or near GABAgated chloride channels. These so called "barbiturate receptors" (9) are allosterically linked to both GABAA and benzodiazepine receptors (9)(10)(11)(12)(13)(14)(15)(16), and this family of allosterically linked recognition sites, the "supramolecular complex" (17), is presumed to mediate the pharmacological actions of many agents that alter neuronal excitability at GABA-gated chloride channels.…”

Section: Introductionmentioning

confidence: 99%

Protection against the lethal effects of pentobarbital in mice by a benzodiazepine receptor inverse agonist, 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline.

Havoundjian¹,

Reed²,

Paul³

et al. 1987

J. Clin. Invest.

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The benzodiazepine receptor inverse agonist 6,7-dimethoxy4-ethyl-3-carbomethoxy-i-carboline (DMCM) (1.5-15 mg/kg) was administered to mice 5 min after a lethal (LD94) injection of pentobarbital. DMCM (1.5-5 mg/kg) increased short-term (1 h) survival in a dose-dependent fashion, with an optimum survival rate more than five times greater than mice receiving pentobarbital alone. Statistically significant increases in longterm (24 h) survival were also observed after both 5 and 10 mg/ kg of DMCM (34 and 33%, respectively) compared with animals receiving pentobarbital alone (6%). Two doses of DMCM (5 and 2.5 mg/kg, respectively) administered 55 min apart produced an even greater increase (58%) in 24-h survival rates. Doses of DMCM that increased 1-and 24-h survival were not lethal when administered alone, and were below the dose that produced convulsions in 50% (CD50) of the animals. The protective effects of DMCM were blocked by pretreatment with the benzodiazepine receptor antagonist ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazoll,5ajll,4]benzodiazodiazepine-3-arboxylate (Ro 15-1788), which suggests the effects of DMCM are mediated through the benzodiazepine receptor. These findings suggest that DMCM or another benzodiazepine receptor ligand with full inverse agonist qualities could prove effective as an antidote for barbiturate intoxication in man.

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Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Cited by 94 publications

References 33 publications

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors

Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes.

Protection against the lethal effects of pentobarbital in mice by a benzodiazepine receptor inverse agonist, 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline.

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Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes

Cited by 94 publications

References 33 publications

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors

Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes.

Protection against the lethal effects of pentobarbital in mice by a benzodiazepine receptor inverse agonist, 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline.

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Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABA_A Receptors