Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [³H]flunitrazepam, [³H]GABA or [³H]muscimol to extensively washed synaptic membranes. Neither the affinity (K_d) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [³H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [³H]flunitrazepam or [³H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA—benzodiazepine receptor ionophore-complex in the brain.     

Effect of picrotoxinin on benzodiazepine receptor binding

The effect of picrotoxinin on [³H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [³H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without alterating binding affinity. In cerebellar membranes, picrotoxinin did not alter [³H]flunitrazepam receptor binding.     

The Purified Gaba/Benzodiazepine/Barbiturate Receptor Complex: Four Types of Ligand-Binding Sites,and the Interactions between them,are Preserved in a Single Isolated Protein Complex

Abstract

A GABA / benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABA_A agonist [³H]muscimol and for the chloride ion channel blocking agent [³⁵S]t-butylbicyclophosphoro-thionate (TBPS), whereas the binding properties for the benzodiazepine [³H] flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53000 and Mr 57000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [³H] flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [³H] flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires > 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments < M_r 10000.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Molecular Interactions of Etazolate with Benzodiazepine and Picrotoxinin Binding Sites

Pyrazolopyridines, such as etazolate (SQ 20009), enhance [3H]diazepam binding to a Lubrol-solubilized fraction that has specific binding sites for 3H benzodiazepines, [alpha-3H]dihydropicrotoxinin (DHP) and [3H]muscimol. Etazolate enhancement of [3H]diazepam binding was inhibited by picrotoxinin. Furthermore, etazolate inhibited the [3H]DHP binding in a Lubrol-solubilized fraction with an IC50 value of 6-8 microM. These results provide evidence that etazolate, like pentobarbital, modulates benzodiazepine binding via the DHP-sensitive site of the benzodiazepine-GABA receptor-ionophore complex.     

GABAA Receptor Subtypes: Ligand Binding Heterogeneity Demonstrated by Photoaffinity Labeling and Autoradiography

Abstract: Heterogeneity of binding affinities for a variety of ligands was observed for γ-aminobutyric acid type A (GABA_A) receptors in the rat CNS, at both GABA and ben-zodiazepine recognition sites. Photoaffinity labeling by [³H]flunitrazepam and [³H]muscimol to affinity column-purified receptor proteins was examined by gel electropho-resis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [³H]flunitrazepam more so into the 51-kDa α1 subunit than into the 53-kDa aL2 polypeptide, and incorporation of [³H]muscimol into the 55-kDa β2 subunit more so than the 58-kDaβ3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine “type 1” selective ligand CL218.872. Heterogeneity in affinity of this drug for the single 51-kDa α1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [³H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THlP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [³H]flunitrazepam binding by steroids, barbiturates, and THlP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isofotms is dependent on the nature of α and subunits other than α, GABA-benzodiazepine coupling is dependent on the nature of the α subunits, GABA site pharmacology is dependent on the nature of the β sub-units, and several subunits including α and β contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.     

Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs

The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Effects of pentylenetetrazol on GABA-A/benzodiazepine/picrotoxinin receptor complexes in rat brain regions

The effects of acute convulsive doses of pentylentetrazol (PTZ) on [³⁵S]t-butyl-bicyclophosphorothionate (TBPS), [³H]flunitrazepam (FNP), [³H]muscimol, and [³H]-aminobutyric acid (GABA) binding sites were examined in well-washed homogenates of various brain regions of rat. Except for a significant increase in the number of striatal [³⁵S]TBPS binding sites, no significant change in [³⁵S]TBPS, [³H]FNP, [³H]muscimol, and [³H]GABA binding was found in various brain regions 30 min after subcutaneous injection of PTZ at 90 or 100 mg/kg. Similarly there were no significant changes in [³⁵S]TBPS and [³H]FNP binding to unwashed P₂ membranes of cerebral cortices 30 min following administration of convulsive doses of PTZ. These experiments failed to demonstrate acute modulation of GABA-A/benzodiazepine/picrotoxinin receptor complex by PTZ in the various brain regions examined except striatum. The significance of the increased [³⁵S]TBPS binding in striatum caused by PTZ remains unclear.     

Human heat shock gene expression and the modulation of plasma membrane Na+, K+-ATPase activity

Benzodiazepine receptor solubilized from bovine cortical membranes was bound to a new benzodiazepine affinity column, the synthesis of which is described. Bio-specific elution with the benzodiazepine compound chlorazepate resulted in the elution of fractions highly enriched in specific binding for the GABA receptor agonist muscimol. Specific activity for [³H]muscimol binding was >1.3 nmol/mg protein. It is shown that [³H]flunitrazepam binding activity can be recovered by removal of chlorazepate from the purified fraction. These results strongly support a model which suggests that the 2 binding sites reside on the same physical entity.     

Differential modulation by muscimol of [3H] flunitrazepam binding to benzodiazepine binding site subtypes

The effects of the GABA agonist, muscimol on [³H]flunitrazepam binding were examined in cerebellum and hippocampus regions proposed to contain different populations of benzodiazepine binding site subtypes. Quantitative analysis was made of the contribution of different components of [³H]flunitrazepam binding by utilising the selective affinities of propyl β-carboline-3-carboxylate for these sites. The influence of muscimol on each of these components was determined and the results provide clear evidence that GABA receptors interact with only some subtypes of benzodiazepine binding sites; for example, whilst the cerebellar site and the low affinity hippocampal site are influenced, the high affinity site in hippocampus appears to be quite unaffected.     

Musciomol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABA_A and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [³H]flunitrazepam and [³H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichement of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays

Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [³H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [³H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Methylmercury modulates GABAA receptor complex differentially in rat cortical and cerebellar membranes in vitro

Effects of methylmercury (MetHg) on the specific [³H]flunitrazepam binding were studied in rat cortical and cerebellar P₂-fractions in vitro. MetHg did not affect significantly the specific [³H]flunitrazepam binding in unwashed P₂-fraction but increased it marginally (by 16%) at 100 M in washed P₂-fraction, in both brain regions.Muscimol (3 M), a GABA_A agonist, stimulated the [³H]flunitrazepam binding by 30% to 50% depending on the brain region. In washed cerebellar membranes the enhancing response of muscimol was 10 to 14% lower after preincubation of the tissue with MetHg but in cerebral cortex MetHg did not modulate the muscimol response at all. The results indicate that Met-Hg may have region specific effects on GABA_A receptors in vitro and the effect may depend on the occupational state of the GABA binding domain of the receptor complex.     

Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain

The effects of acute and chronic administration of a subconvulsive dose of picrotoxin on t-[35S]butylbicyclophosphorothionate ([3S]TBPS), [3H]muscimol, and [3H]flunitrazepam binding characteristics in various regions and on the convulsant potency of picrotoxin in Sprague-Dawley rats were examined. Acute administration of a subconvulsive dose of picrotoxin (3 mg/kg, i.p.) significantly increased [35S]TBPS and [3H]muscimol binding in cerebellum (CB) with no change in frontal cortex (FC). In rats treated chronically with picrotoxin (3 mg/kg, i.p., daily for 10 days), the Bmax of [35S]TBPS binding site was significantly decreased in the FC, striatum (ST), and CB with no change in KD values. Neither [3H]muscimol binding in the FC and CB nor [3H]flunitrazepam binding in the FC was affected in these rats. In addition, the potency of pentobarbital to inhibit [35S]TBPS binding in vitro was not altered following acute or chronic treatment of picrotoxin. Chronic administration of picrotoxin did not affect convulsive ED50 or LD50 of picrotoxin; however, it delayed the onset of convulsions and increased the time to death. These results suggest that treatment with picrotoxin at a subconvulsive dose for 10 days causes down-regulation of [35S]TBPS binding sites and that this down-regulation might be related, at least in part, to the decreased extent of convulsant potency of picrotoxin. In addition, the results indicate possible interaction between convulsant binding sites and GABAA receptor sites in the CB following picrotoxin treatment.     

Effects of chronic ethanol inhalation on the enhancement of benzodiazepine binding to mouse brain membranes by GABA

Chronic ethanol inhalation produced no change in the number or affinity of [³H]flunitrazepam binding sites on well-washed synaptic membranes prepared from male Quackenbush mice, but produced a significant decrease in the capacity of GABA to enhance [³H]flunitrazepam binding. This decrease was characterised by a higher EC₅₀ (1.4 μ M compared to 0.6 μ M) and a lower maximal level of enhancement (162% compared to 172%) for tissue from the chronically treated animals compared to tissue from control animals. Acute ethanol treatment or ethanol incubated in vitro with the brain membranes did not produce changes in any of the [³H]flunitrazepam binding parameters. These results support other findings that chronic ethanol may affect the coupling of various sites on GABA-A receptor-ionophore complexes in brain.     

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)