t-[35S]Butylbicyclophosphorothionate Binding under Equilibrium and Nonequilibrium Conditions: Differential Effects of Barbiturates and γ-Aminobutyric Acid in the Long-Sleep and Short-Sleep Selected Mouse Lines

Significant differences were demonstrated between the long-sleep (LS) and short-sleep (SS) selected mouse lines in the abilities of barbiturates and gamma-aminobutyric acid (GABA) to inhibit t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to well-washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor-chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.     

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.     

Gamma-aminobutyric acid and alcohol actions: neurochemical studies of long sleep and short sleep mice

Effects of ethanol and pentobarbital on the GABA receptorchloride channel complex were evaluated in mice selected for differential sensitivity to the hypnotic effects of ethanol (long sleep and short sleep lines). 36Cl- influx, [35S]tbutylbicyclophosphorothionate (TBPS) and [3H]muscimol binding were measured in a membrane vesicle suspension (microsacs) from cerebellum or forebrain. Muscimol was found to be a more potent stimulator of 36Cl- flux in the LS cerebellum, as compared to the SS cerebellum, but a similar maximal level of uptake was achieved in the two lines. Muscimol displaced [35S]TBPS (a ligand for the convulsant site) from cerebellar microsacs, and LS mice were also more sensitive than SS mice to this action of muscimol. However, the number or affinity of high affinity [3H]muscimol binding sites did not differ between the lines. Physiologically relevant concentrations of ethanol (15-50 mM) potentiated muscimol stimulation of 36Cl- uptake in LS cerebellum but had no effect in SS cerebellum. Ethanol failed to alter stimulated chloride flux hippocampal microsacs from either line. Both the LS and SS lines responded similarly to pentobarbital potentiation of muscimol stimulated chloride uptake regardless of brain region. The demonstrated difference between the LS and SS mice in muscimol stimulated chloride uptake as well as in muscimol displacement of [35S]TBPS binding offers a biochemical explanation for the line differences in behavioral responses to GABAergic agents. Moreover, the findings suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of GABA-operated chloride channels to ethanol.     

Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.     

Protection of soluble benzodiazepine receptors from heat inactivation by GABAergic ligands

Benzodiazepine receptors were solubilized from calf brain cortex by the ionic detergent deoxycholate and by the nonionic detergent Triton X-100. Approximately 90% of the soluble benzodiazepine receptors of both preparations were heat inactivated within 30 min at 55 degrees C. 100 microM of gamma-aminobutyric acid (GABA) protected 80% of Triton X-100 solubilized benzodiazepine receptors and 56% of the deoxycholate soluble benzodiazepine receptors from heat inactivation. Time course of heat inactivation showed that the deoxycholate soluble receptors are more sensitive to heat than the Triton X-100 soluble receptors.     

Modulation of γ-Aminobutyric AcidA Receptor-Operated Chloride Channels by Benzodiazepine Inverse Agonists Is Related to Genetic Differences in Ethanol Withdrawal Seizure Severity

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of γ-Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors

We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.     

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Effect of benzodiazepines on NAD binding to synaptic membrane in the rat brain

The receptor protein solubilized from synaptic membranes specifically binds [14C] NAD (dissociation constant--0.75 microM, capacity of binding sites--0.0125 nmol of metaid per 1 mg of protein). All the studied benzodiazepines (phenazepam, nitrazepam, clonazepam, flunitrazepam) are able to displace [14C] NAD from its receptor sites, the mixed type of inhibition being manifested. An inhibition constant for flunitrazepam, a ligand of benzodiazepine receptors, equals 10 microM. GABA promotes an inhibiting effect of benzodiazepines. It is supposed that neurotropic action of NAD is realized through the GABA-benzodiazepine complex of neuronal membranes.     

Radiation Inactivation Studies of the Benzodiazepine/γ-Aminobutyric Acid/Chloride Ionophore Receptor Complex

Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), gamma-aminobutyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15-1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl-beta-carboline-3-carboxylate) averaged 51,000 +/- 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t-butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at -135 degrees C or 30 degrees C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30 degrees C, the target size for beta-galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose-6-phosphate dehydrogenase was not altered by lyophilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.     

Solubilisation of the γ-Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids

We have solubilised the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3-[(3-cholamidopropyl)dimethylammonio] 1-propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 +/- 0.2 nM compared to a KD of 2.3 +/- 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4 degrees C. This is contrary to the results obtained with a CHAPS-soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.     

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)