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1.
Previous studies have shown that ethanol may produce some of its effects by facilitation of GABAergic transmission. One of the potential sites of drug action at the GABA receptor complex is the picrotoxin site, which can be studied with [35S]t-butylbicyclophosphorothionate (TBPS). Ethanol inhibited the binding of [35S]TBPS to C57 mice brain regions in vitro. This inhibition appears to be noncompetitive since ethanol decreased the Bmax and not the KD value of [35S]TBPS. C57 mice were chronically treated with ethanol in liquid diet to determine if the sensitivity of TBPS binding is altered following chronic treatment or during withdrawal. Chronic treatment with ethanol and during withdrawal did not alter the KD or Bmax values of [35S]TBPS binding in C57 mice brain regions. It is suggested that the sensitivity of picrotoxin site on the oligomeric GABA receptor complex is not altered during ethanol tolerance or withdrawal. The effects of ethanol on GABA system may be mediated by its interaction with the coupling mechanism(s) or a direct effect on the chloride channels.  相似文献   

2.
We searched for subunit correlations for GABA(A) receptor-associated atypically GABA-insensitive [35S]TBPS binding. The homomeric beta3 subunit receptors could be excluded, as GABA-insensitive [35S]TBPS binding was present in beta3-/- mice. Localization of GABA-insensitive [35S]TBPS binding correlated best with those of delta, alpha4 and alpha6 subunit mRNAs. The amounts of GABA-insensitive [35S]TBPS binding components were increased in delta-/- mice, but dramatically reduced in alpha6-/- mice, suggesting a role for alpha6 but excluding delta subunits.  相似文献   

3.
The epilepsy that occurs in SSADH deficiency has a seizure phenotype similar to that occurring in the SSADH(-/-) mouse. We examined the expression and function of the GABA(A) receptor (GABA(A)R) in SSADH-deficient mice. A selective decrease in binding of [(35)S]tert-butylbicyclophosphorothionate was observed in SSADH(-/-) mice at postnatal day 7 that was progressive until the third postnatal week of life when, at the nadir of the decreased [(35)S]tert-butylbicyclophosphorothionate binding, generalized convulsive seizures emerged that rapidly evolved into status epilepticus. We also observed a substantial downregulation of the beta(2) subunit of GABA(A)R, a reduction in GABA(A)-mediated inhibitory postsynaptic potentials, and augmented postsynaptic population spikes recorded from hippocampal slices. The SSADH(-/-) mouse model represents a powerful investigative tool for understanding the pathophysiology of the seizures associated with human SSADH deficiency. These data raise the possibility that progressive dysfunction of the GABA(A)R may be involved in the development of seizures in SSDAH-deficient mice. Elucidation of the precise fundamental mechanisms of the perturbation of the GABA(A)R-mediated function in SSADH(-/-) mice could lead to the development of novel treatment modalities designed to reduce the neurological morbidity in children with SSADH deficiency.  相似文献   

4.
t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
The gamma-aminobutyric acid (GABA) receptor-operated chloride channel complex was evaluated in mice selected for differential sensitivity to the ataxic effects of diazepam (diazepam-sensitive (DS) and diazepam-resistant (DR) lines). The ataxic effects of several drugs purported to produce some of their actions through the benzodiazepine-GABA receptor complex were examined using the rotarod test. The duration of impairment produced by diazepam, ethanol, 4,5,6,7-tetrahydroisoxazol[5,4-C]pyridine-3-ol (THIP) and phenobarbital was greater in the diazepam-sensitive than in the diazepam-resistant mice. In contrast, pentobarbital produced an equivalent duration of ataxia in the two lines. Muscimol-stimulated 36Cl- influx and the binding of [35S]t-butylbicyclophosphorothionate (TBPS) and [3H]flunitrazepam were measured using isolated brain membrane vesicles (microsacs). Depolarization-dependent 45Ca2+ uptake was measured in whole brain synaptosomes. Muscimol was a more potent stimulator of 36Cl- flux in the DS compared to the DR mice, although no difference between the lines was found in muscimol-stimulation of [3H]flunitrazepam binding. Flunitrazepam augmented the muscimol-stimulated 36Cl- uptake in the DS but not in the DR mice. However, no differences between the lines of mice were found in either density or affinity of [3H]flunitrazepam binding sites. Similarly, no differences in either the density or affinity of [35S]TBPS binding sites was found. Ethanol (10-45 mM) potentiated the muscimol-stimulation of 36Cl- in DS, with no effect in DR mice. However, ethanol inhibition of [35S]TBPS binding was equivalent in the two lines of mice. Pentobarbital produced an equal potentiation of the muscimol-stimulated 36Cl- flux in the two lines, but phenobarbital potentiated the muscimol-induced 36Cl- influx slightly more in DS mice.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
t- Butylbicyclophosphorothionate ( TBPS ), a derivative of potent GABA antagonistic cage convulsants, has recently been introduced ( Squires , R. F., J.E. Casida , M. Richardson, and E. Saederup (1983) Mol. Pharmacol. 13:326-336) as ligand for a GABA-A receptor-linked drug receptor. Using conventionally prepared washed membrane fractions from rat cerebral cortex, we have confirmed that in the presence of 200 mM NaBr [35S] TBPS binds to a high affinity population of binding sites (Kd 26 +/- 5 nM) and that muscimol inhibits [35S] TBPS binding (IC50 0.32 microM) allosterically. In 200 mM NaCl the apparent affinity of [35S] TBPS binding sites is lower (Kd 60 +/- 5 nM), and muscimol has biphasic effects with stimulation at low concentrations of muscimol (EC50 0.023 microM) followed by inhibition at high concentrations (IC50 0.72 microM). Both base line [35S] TBPS binding (in 200 mM NaCl) and muscimol inhibition of [35S] TBPS binding (in 200 mM NaBr) are bidirectionally modulated by the occupancy of benzodiazepine receptors with its ligands. Benzodiazepine receptor agonists, regardless of their structure, enhance and inverse benzodiazepine receptor agonists inhibit base line [35S] TBPS binding and muscimol inhibition of [35S] TBPS binding. Fourteen ligands for benzodiazepine receptors display a similar in vitro profile as benzodiazepine receptor agonists or inverse benzodiazepine receptor agonists on [35S] TBPS binding as their anti- or proconvulsive effects in vivo suggest (Jensen, L. H., E. N. Petersen, and C. Braestrup (1983) Life Sci. 33: 393-399). That [35S] TBPS binding sites are constituents of a GABA benzodiazepine receptor complex is also suggested by a number of membrane pretreatments.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
Human succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of GABA metabolism associated with motor impairment and epileptic seizures. Similarly, mice with targeted deletion of the Aldh5a1 gene (Aldh5a1–/–) exhibit SSADH deficiency and seizures early in life. These seizures begin as absence seizures the second week of life, but evolve into generalized convulsive seizures that increase in severity and become lethal during the fourth postnatal week. The seizures are alleviated and survival is prolonged when the mutant animals are weaned onto a ketogenic diet (KD). The persistence of spontaneous, recurrent, generalized tonic–clonic seizures in KD-treated adult Aldh5a1–/– mice allowed us to quantify their daily (circadian) distribution using a novel behavioral method based on the detection of changes in movement velocity. Adult KD-treated Aldh5a1–/– mice exhibited a seizure phenotype characterized by fits of wild running clonus accompanied by jumping and bouncing. These hypermotor seizures were largely spontaneous and occurred daily in a nonrandom pattern. The seizure rhythm showed a peak shortly after dark phase onset (2008 hours) with near-24-hour periodicity. Age-matched wild-type littermates showed no evidence of abnormal motor behavior. These new data suggest that generalized tonic–clonic seizures in Aldh5a1–/– mice are more frequent during a specific time of day and will provide useful information to clinicians for the treatment of seizures associated with human SSADH deficiency.  相似文献   

8.
The succinic semialdehyde dehydrogenase (SSADH) null mouse (SSADH(-/-)) represents a viable animal model for human SSADH deficiency and is characterized by markedly elevated levels of both gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acid (GABA) in brain, blood, and urine. In physiological concentrations, GHB acts at the GHB receptor (GHBR), but in high concentrations such as those observed in the brains of children with SSADH deficiency, GHB is thought to be a direct agonist at the GABABR receptor (GABABR). We tested the hypothesis that both GHBR and GABABR-mediated function are perturbed in SSADH deficiency. Therefore, we examined the high affinity binding site for GHB as well as the expression and function of the GABABR in mutant mice made deficient in SSADH (SSADH(-/-)). There was a significant decrease in binding of the specific GABABR antagonist, [3H]CGP-54626A at postnatal day (PN)7 and PN14 in SSADH(-/-) when compared to wild type control animals (SSADH(+/+)), particularly in hippocampus. GABABR-mediated synaptic potentials were decreased in SSADH(-/-). Immunoblot analysis of GABABR1a, R1b, and R2 in SSADH(-/-) indicated a trend towards a region-specific and time-dependent decrease of GABABR subunit protein expression. There was no difference between SSADH(-/-) and wild type in binding of either [3H]GHB or a specific GHBR antagonist to the GHBR. These data suggest that the elevated levels of GABA and GHB that occur in SSADH(-/-) lead to a use-dependent decrease in GABABR-mediated function and raise the possibility that this GHB- and GABA-induced perturbation of GABABR could play a role in the pathogenesis of the seizures and mental retardation observed in SSADH deficiency.  相似文献   

9.
In rats, the degeneration of the striato-nigral GABAergic pathway caused by the intrastriatal injection of kainic acid induced a marked decrease (65%) of GABA content and glutamic acid decarboxylase (GAD) activity and a dramatic increase (225%) in the binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a membrane preparation from the substantia nigra homolateral to the injected striatum. The increase in [35S]TBPS binding in the denervated substantia nigra was exclusively due to an increased density of binding sites (Bmax) with no change in the dissociation constant (kd). The enhancement in [35S]TBPS binding was almost completely reversed by the intraperitoneal administration of diazepam (3 mg/kg) to kainic acid-lesioned rats. Moreover, diazepam produced a significant decrease (30%) in the density of [35S]TBPS binding sites also in the sham-operated side. In contrast the 'in vitro' addition of the GABAA receptor antagonist bicuculline (1 microM) to the membrane preparation from the denervated substantia nigra further increased [35S]TBPS binding. These findings suggest the view that the increase of nigral [35S]TBPS binding is directly related to the inhibition in the function of nigral GABAergic synapses following the loss of the striato-nigral GABAergic pathway. Our results indicate that [35S]TBPS binding to brain structure is a potential tool to reveal alteration in the function of GABAA receptor complex elicited by physiological, pharmacological and pathological conditions.  相似文献   

10.
Gamma-ainobutyric acid type A (GABA(A)) receptor ionophore ligand t-[35S]butylbicyclophosphorothionate ([35S]TBPS) was used in an autoradiographic assay on brain cryostat sections to visualize and characterize atypical GABA-insensitive [35S]TBPS binding previously described in certain recombinant GABA(A) receptors and the cerebellar granule cell layer. Picrotoxinin-sensitive but 1-mM GABA-insensitive [35S]TBPS binding was present in the rat cerebellar granule cell layer, many thalamic nuclei, subiculum and the internal rim of the cerebral cortex, amounting in these regions up to 6% of the basal binding determined in the absence of exogenous GABA. Similar binding properties were detected also in human and chicken brain sections. Like the GABA-sensitive [35S]TBPS binding, GABA-insensitive binding was profoundly decreased by pentobarbital, pregnanolone, loreclezole and Mg2+. The binding was reversible and apparently dependent on Cl- ions. Localization of the GABA-insensitive [35S]TBPS binding was not identical to that of high-affinity [3H]muscimol binding and diazepam-insensitive [3H]Ro 15-4513 binding, two previously established receptor subtype-dependent binding heterogeneities in the rat brain. The present study reveals a component of the GABA-ionophore enriched in the thalamus and cerebellar granule cells, possibly representing poorly desensitized or desensitizing receptors.  相似文献   

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