TBPS Research Articles

Effects of the conformationally restricted GABA analogues, cis- and trans-4-aminocrotonic acid, on GABA neurotransmission in primary neuronal cultures.

The effects of the GABA analogues, cis- and trans-4-aminocrotonic acid (ACA) on GABA(A) receptor function and GABA uptake, together with the presence of p-1 subunit mRNA and putative GABAc receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl- influx, which was inhibited by bicuculline, t-butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and gamma-hexachlorocyclohexane (gamma-HCH or lindane). [3H]-flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the transisomer completely inhibited the [3H]GABA uptake, whereas the cis-isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABAc receptors was investigated only in neocortical cultures by using RT-PCR in order to detect the presence of the mRNA encoding the p-1 subunit which assembles to form homooligomeric Cl-channels. The results presented here show that p-1 subunits, and thus GABAc receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA(A) receptors, although with very different potencies.

The Interaction of Chlorinated Alicyclic Insecticides with Brain GabaA Receptors in Channel Catfish (Ictalurus Punctatus)

Chlorinated alicyclic insecticides are believed to antagonize the action of the neurotransmitter γ-aminobutyric acid (GABA) at its receptor in vertebrates. Binding of the specific GABAA receptor ligand [35S]-t-butylbicyclophosphorothionate (TBPS) to channel catfish brain P2 membranes suggested a single population of receptors with a Kd (56.6 ± 2.6 nM) and Bmax (2435 ± 276 fmol/mg protein) that are similar to published values for other fish species. The competition of several chlorinated compounds for TBPS binding was investigated. The most potent inhibitors of TBPS binding were 12-ketoendrin, photoheptachlor epoxide, photoheptachlor, telodrin, and endrin, respectively, with IC50s of 20-90 nM. Photooxychlordane, photo α-chlordane, and oxychlordane were intermediate in potency (122-219 nM), as were isodrin, dihydroisodrin, heptachlor epoxide, and α-chlordane, which were similar in potency (311-397 nM). Dieldrin, lindane, and dihydroaldrin were much less potent (592-1103 nM). Heptachlor, aldrin, and γ-chlordane were weak inhibitors of TBPS binding (2073-2738 nM). Chlordene and chlordecone had the lowest potency of all compounds studied (10, 201-21, 178 nM) with the exception of mirex, which did not inhibit binding at a concentration of 50 μM. There is a good correlation between binding potency and the available toxicity data for several of these compounds in channel catfish. There is also a good correlation between the inhibitory potency in channel catfish by these types of compounds with that in rats.

Honokiol and magnolol increase the number of [3H] muscimol binding sites three-fold in rat forebrain membranes in vitro using a filtration assay, by allosterically increasing the affinities of low-affinity sites.

1. The bark of the root and stem of various Magnolia species has been used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. The biphenolic compounds honokiol (H) and magnolol (M), the main components of the Chinese medicinal plant Magnolia officinalis, interact with GABA(A) receptors in rat brain in vitro. We compared the effects of H and M on [3H]muscimol (MUS) and [3H]flunitrazepam (FNM) binding using EDTA/water dialyzed rat brain membranes in a buffer containing 150 mM NaCl plus 5 mM Tris-HCl, pH 7.5 as well as [35S]t-butylbicyclophosphorothionate (TBPS) in 200 mM KBr plus 5 mM Tris-HCl, pH 7.5. H and M had similar enhancing effects on [3H]MUS as well as on [3H]FNM binding to rat brain membrane preparations, but H was 2.5 to 5.2 times more potent than M. 2. [3H]FNM binding. GABA alone almost doubled [3H]FNM binding with EC50 = 450 nM and 200 nM using forebrain and cerebellar membranes, respectively. In the presence of 5 microM H or M the EC50 values for GABA were decreased to 79 and 89 nM, respectively, using forebrain, and 39 and 78 nM, using cerebellar membranes. H and M potently enhanced the potentiating effect of 200 nM GABA on [3H]FNM binding with EC50 values of 0.61 microM and 1.6 microM using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 microM, and 22 and 34%. 3. [3H]MUS binding. H and M increased [3H]MUS binding to whole forebrain membranes about 3-fold with EC50 values of 6.0 and 15 microM. Using cerebellar membranes, H and M increased [3H]MUS binding approximately 68% with EC50 values of 2.3 and 12 microM, respectively. Scatchard analysis revealed that the enhancements of [3H]MUS binding were due primarily to increases in the number of binding sites (Bmax values) with no effect on the high affinity binding constants (Kd values). The enhancing effect of H and M were not additive. 4. [35S]TBPS binding. H and M displaced [35S]TBPS binding from sites on whole rat forebrain membranes with IC50 values of 7.8 and 6.0 microM, respectively. Using cerebellar membranes, the corresponding IC50 values were 5.3 and 4.8 microM. These inhibitory effects were reversed by the potent GABA(A) receptor blocker R5135 (10 nM), suggesting that H and M allosterically increase the affinity of GABA(A) receptors for GABA and MUS by binding to sites in GABA(A) receptor complexes. 5. Two monophenols, the anesthetic propofol (2,6-diisopropylphenol, P) and the anti-inflammatory diflunisal (2',4'-difluoro-4-hydroxy-3-biphenyl carboxylic acid, D) also enhanced [3H]MUS binding, decreased the EC50 values for GABA in enhancing [3H]FNM binding and potentiated the enhancing effect of 200 nM GABA on [3H]FNM binding, although enhancements of [3H]MUS binding for these monophenols were smaller than those for H and M, using forebrain and cerebellar membranes. The enhancing effect of P and D on [3H]MUS binding were almost completely additive. 2,2'-biphenol was inactive on [3H]MUS and [3H]FNM binding. These, and other preliminary experiments, suggest that appropriate ortho (C2) and para (C4) substitution increases the GABA-potentiating activity of phenols. 6. The potentiation of GABAergic neurotransmission by H and M is probably involved in their previously reported anxiolytic and central depressant effects.

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABA A receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ 3 H ]flunitrazepam (2 nM), [ 3 H ]muscimol (5 nM), and 4 nM [ 35 S ] t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ 3 H ]flunitrazepam and [ 3 H ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ 3 H ]flunitrazepam as well as [ 3 H ]muscimol binding ( E max) in the ethanol-dependent rat cerebellum as compared to control group ( p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ 35 S ]TBPS at lower concentrations (⩽100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ 35 S ]TBPS binding (− E max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats ( p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid A (GABA A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.

Caloric restriction retards the aging associated changes in γ-aminobutyric acid A receptor gene expression in rat cerebellum

It is widely accepted that calorie restriction is an effective way of delaying the aging process. Also, there is an indication that the beneficial effects exerted by dietary manipulation may be due to a direct effect at the molecular level like gene expression. The studies were conducted to determine whether calorie restriction prevents any age-related changes in the structural and molecular aspects of the GABA A–BZ receptor. In aged (24-month old diet ad libitum) rats, the binding of [ 35S] t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced in the cerebellum. In contrast, [ 35S]TBPS binding remained unchanged in the cerebellum of calorie restricted old rats. In order to evaluate the molecular basis of these changes, the α sub-unit mRNA levels were measured. The GABA A receptor α 1 sub-unit mRNA level remained unchanged in both the old groups of rats. The α 2 subunit mRNA level was significantly decreased in the cerebellum of aged rats (24-month old ad libitum), whereas it remained unchanged in the cerebellum of calorie restricted old animals. These findings indicate a selective age and diet related modulation in the stoichiometry of the GABA A receptor in aging.

Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures.

The single-locus mutant mouse tottering (tg) is an established model for absence seizures. We have previously reported an impairment in GABA-induced chloride uptake in tg brain [Tehrani and Barnes, Epilepsy Res. 1995;22:13-21]. In order to determine if this alteration in GABAA receptor function can be related to specific receptor isoforms, we examined the radioligand binding properties of GABAA receptors and the expression of GABAA receptor subunit mRNAs in the cerebral cortex. Saturation binding of [3H]flunitrazepam revealed a significantly lower Kd value in tg cortical tissues (1.77 +/- 0.05 nM) in comparison to that for the background C57BL/6J strain (3.23 +/- 0.23 nM), while the Bmax values were indistinguishable. Biphasic displacement of [3H]flunitrazepam binding by 2-oxoquazepam showed that low affinity binding sites account for 36 +/- 7.6 and 51 +/- 7.5% of the total in control and tg, respectively. The level of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to tg cortical membranes was 73.6 +/- 5.8% of that in controls. Paired measurements by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant differences in the levels of GABAA receptor alpha 1, alpha 3, alpha 5, beta 2, beta 3, gamma 2 or gamma 3 subunit mRNAs between tg and control cortex. However, tg tissues showed elevated levels of alpha 2- and beta 1-subunit mRNAs, representing 256 and 177%, respectively, those of controls. For the tg cortex, the enhanced expression of GABAA receptor alpha 2 and beta 1 subunits correlates with recombinant subtypes known to have low affinity for 2-oxoquazepam and impaired binding of TBPS. These aberrant properties of GABAA receptors could influence the development or propagation of phenotypic seizures in the tottering mouse.

Role of NMDA receptors in pentobarbital tolerance/dependence.

Effects of continuous pentobarbital administration on binding characteristics of [3H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300 micrograms/10 microliters/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [3H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10 microliters/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [35S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.

Arachidonic acid and eicosanoids in Hydra: possible endogenous signals involved in chemoreception and modulation of the feeding behaviour.

Arachidonic acid (AA) exerts many biological actions in different tissues. Modulation of ion channels by AA has received considerable attention in studies of the role of this fatty acid in the nervous system. In mammalian central nervous system polyunsaturated fatty acids (PUFAs) have been found to regulate the activity of GABAA receptor-coupled Cl− channels1, 2. AA serves also as a precursor of a variety of eicosanoids, among which lipoxygenase metabolites with R stereochemistry have been identified in several species of marine and freshwater invertebrates3. AA and its ω10-lipoxygenase metabolites, as well as phospholipase A2 (PLA2), the enzyme mostly responsible for PUFA release from membrane phospholipids have been found in Hydra 3, one of the most primitive organisms to evolve a nervous system4. The freshwater polyp Hydra is a predator whose tentacles have stinging capsules named nematocysts; when the prey touches a tentacle a complex behavioral phenomenon, the feeding response, is initiated by the outflow of reduced glutathione (GSH) from the wounded prey5. This response consists of a sequence of events, including tentacle writhing, mouth opening, ingestion of prey and closing of the mouth. Duration of response, i. e. the interval between mouth opening (Ti) and mouth closure (Tf) time is dependent on GSH concentration through the interaction with an external receptor6 and can be quantitatively measured. At present, mechanisms of termination of the response are still poorly understood, though we have recently shown that γ-aminobutyric acid (GABA) prolongs the duration of response through a receptor-mediated action7. Further biochemical and physiological studies have shown that neuroactive steroids, benzodiazepines, bicuculline, picrotoxin and the specific Cl− channel ligand t-butylbicyclophosphorothionate (TBPS) also modulate response duration, suggesting that a population of GABAA-like receptors and/or GABA-gated Cl− channels are involved8. Here we present the results of an in vivo study on the participation of AA and some of its metabolites in the GSH-induced feeding response in Hydra.

Characterization of the furanocoumarin phellopterin as a rat brain benzodiazepine receptor partial agonist in vitro

Phellopterin, a naturally occurring furanocoumarin found in the roots of Angelica dahurica, inhibits [ 3H]diazepam and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([ 3H]Ro 15-1788) binding to the benzodiazepine site of the rat brain γ-aminobutyric acid A (GABA A) receptor in vitro with IC 50 values of 400 and 680 nM, respectively. Two other naturally occurring furanocoumarins, byakangelicol and imperatorin were significantly less potent, with IC 50 values for inhibition of [ 3H]diazepam binding of 8.0 and 12.3 μM, respectively. Scatchard plot analysis showed that the inhibitory activity of phellopterin was due to competitive inhibition of the benzodiazepine ligand binding. The results of GABA- and t-butylbicyclophosphorothionate (TBPS)-shift assays suggest that phellopterin is a partial agonist of the central benzodiazepine receptors in vitro.

Astroglia-released factor with negative allosteric modulatory properties at the GABA A receptor : Evidence from binding studies

We have previously shown, using whole-cell patch-clamp techniques, that astrocytes release a negative allosteric modulator of the γ-aminobutyric acid type A receptor (GABA A receptor) with β-carboline-like properties, thus, likely to act at the benzodiazepine site. Here, using patch-clamp and binding techniques, we confirm that the low-molecular-weight fraction of astroglia-conditioned medium (ACM lmf) contains a factor(s) that negatively modulates GABA A-receptor function. This factor, like β-carbolines, enhances the specific binding of [ 35S] t-butyl bicyclophosphorothionate (TBPS) to adult rat cortical membranes in the presence of GABA. However, it fails to interact with various ligands of the benzodiazepine (BZD) site of the GABA A receptor ([ 3H]flunitrazepam, [ 3H]Ro 15-1788 and [ 3H]Ro 15-4513). The question of the actual binding site of the astroglia-derived factor on the GABA A receptor, thus, remains open and can be addressed only after the purification of the active molecule(s) of ACM Imf has been completed, and a labeled form of the endogenous ligand becomes available. Taken together, however, the data suggest that type 1 astrocytes are able to modulate the effects of the main inhibitory neurotransmission in the central nervous system.

Effect of ACTH on resynthesis of GABA transaminase and GABAa receptor complex in rat brain

Adrenocorticotrophic hormone (ACTH) has clinical efficacy for some neurologic disorders, especially seizure disorders of childhood such as infantile spasms. In order to investigate the anticonvulsant mechanism of ACTH, the effect of ACTH1-24 injection on resynthesis of GABA transaminase (GABA-T) was investigated by enzyme histochemical techniques after systemic administration of gabaculine, an irreversible inhibitor of GABA-T. The effects of ACTH1-24 were also investigated on GAB-stimulated 36 Cl − uptake, [ 35 S] t -butylbicyclophosphorothionate (TBPS) binding and on [ 3 H]GABA binding in rat cerebrocortical membranes in vitro. Daily injection of ACTH1-24 accelerates resynthesis of GABA-T activity, suggesting the anticonvulsant potency of this compound. On the other hand, ACTH1-24 showed a dose-related inhibition of net uptake of 36 Cl − , and at higher concentrations, showed a statistically significant inhibitory effect on [ 3 H]GABA binding. ACTH1-24 also showed a significant dose-related enhanced effect on [ 35 S]TBPS binding in the presence of 1 μM exogenous GABA. These results demonstrate rather an antagonistic effect on GABA a receptor, which may correlate with the potential proconvulsant activity.

Interaction of allosteric ligands with GABA A receptors containing one, two, or three different subunits

The presence of allosteric binding sites on recombinant GABA A receptors formed after transfection of human embryonic kidney (HEK) 293 cells with α 1-, β 3-, γ 2-subunits, or with various combinations of these subunits, was systematically investigated. From all possible subunit combinations, high affinity [ 3H]muscimol binding sites were induced in cells transfected with α 1 β 3- or α 1 β 3 γ 2-subunits only. GABA A receptor associated [ 3H]flunitrazepam binding sites were induced in cells after transfection with α 1 γ 2- or α 1 β 3 γ 2-subunits, and [ 35S] t-butylbicyclophosphorothionate (TBPS) binding sites were found in cells transfected with β 3-, β 3 γ 2-, α 1 β 3-, or α 1 β 3 γ 2-subunits. Binding of [ 35S]TBPS could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and 4′-chlorodiazepam (Ro 5-4864) with a potency which differed in cells transfected with β 3-, β 3 γ 2-, α 1 β 3-, or α 1 β 3 γ 2-subunits. Results obtained indicate that receptors with different subunit composition actually can be formed in HEK cells and exhibit distinct pharmacological properties.

Extensive Heterogeneity of Recombinant γ-Aminobutyric AcidA Receptors Expressed in α4β3γ2-Transfected Human Embryonic Kidney 293 Cells

Human embryonic kidney 293 cells transiently transfected with α4-, β3- and γ2-subunits of γ-aminobutyric acidA (GABAA) receptors from the rat exhibited specific high affinity binding sites for [3H]muscimol, [3H]Ro 15-4513 and [35S]t-butylbicyclophosphorothionate (TBPS). Bmax values obtained, however, were dramatically different for these compounds. In addition, GABA was able to inhibit only 20% of specific [35S]TBPS binding to membranes from α4β3γ2-transfected cells. In order to investigate possible receptor heterogeneity, receptors were extracted from α4β3γ2-transfected cells and were fractionated by chromatography on an anti-γ2-, followed by an anti-α4- and an anti-β3-immunoaffinity column. Western blot analysis of the column eluates indicated the separate existence of GABAA receptors consisting of α4β3γ2-, α4β3- or β3-subunits in α4β3γ2-transfected cells. This, and the finding that only α4β3γ2- but not α4β3- or β3-receptors possess high affinity binding sites for all three radiolabeled ligands investigated, combined with the observation that [35S]TBPS binding to receptors consisting of β3-subunits cannot be inhibited by GABA, can explain most of the binding data obtained. The present results suggest an inefficient assembly of γ2- with α4- and/or β3- subunits under the conditions used, and indicate that recombinant receptors expressed in HEK cells are not necessarily homogeneous. Copyright © 1996 Elsevier Science Ltd

The Anaesthetic Action and Modulation of GABAA Receptor Activity by the Novel Water-soluble Aminosteroid Org 20599

The anaesthetic profile of a novel water-soluble aminosteroid, Org 20599 [(2β,3α,5α)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one methanesulphonate], and the ability of the compound to allosterically regulate the activity of the GABAA receptor, have been studied in comparison to the properties of established intravenous general-anaesthetic agents. Intravenously administered Org 20599 produced a rapid onset, short duration loss of the righting reflex in mice. The anaesthetic potency of Org 20599 was comparable to that of the steroids 5α-pregnan-3α-ol-20-one or alphaxalone, and exceeded that of propofol, thiopentone or pentobarbitone. Org 20599 and the reference anaesthetic agents allosterically displaced the binding of [35S]-t-butylbicyclophosphorothionate (TBPS) from GABAA receptors of rat-brain membranes with the order of potency: 5α-pregan-3α-ol-20-one > Org 20599 > alphaxalone > propofol > thiopentone > pentobarbitone. At human recombinant α1, β2, γ2L subunit-containing GABAA receptors expressed in Xenopus laevis oocytes, the anaesthetic agents produced a concentration-dependent and reversible potentiation of the peak amplitude of GABA-evoked currents. A similar positive allosteric action of Org 20599 was observed for the GABAA receptors expressed by bovine adrenal chromaffin cells maintained in culture. The rank order of potency in the aforementioned assays was identical to that determined from the displacement of TBPS binding. At concentrations greater than those required for potentiation of GABA, the anaesthetics exhibited GABA-mimetic effects with a rank order of potency that paralleled their modulatory activity. Such direct agonism varied greatly in maximal effect between compounds. The modulatory and direct agonist actions of Org 20599 were additionally confirmed utilizing rat hippocampal neurones in culture. The results indicate Org 20599 to be a potent and short-acting intravenous anaesthetic agent in mice and suggest positive allosteric regulation of GABAA receptor function to be a plausible molecular mechanism of action for the drug. Copyright © 1996 Elsevier Science Ltd.

Taurine allosterically inhibits binding of [ 35S]- t-Butylbicyclophosphorothionate (TBPS) to rat brain synaptic membranes

The modulatory effects of taurine on [ 35S]- t-Butylbicyclophosphorothionate (TBPS) binding to rat brain synaptic membranes were evaluated and compared with that of GABA. Taurine allosterically inhibited TBPS binding by interacting with a bicuculline-sensitive site, similar to GABA. Taurine was as effective as GABA but less potent. The potency of taurine inhibition of TBPS binding varied among brain regions with cerebellum > olfactory bulb > cortex, similar to that of GABA. Inhibition of TBPS binding to cortical membranes measured under nonequilibrium conditions yielded a dynamic biphasic inhibition curve that was similarly shaped for GABA and taurine. The effect of taurine on TBPS binding was pharmacologically specific in that β-alanine and guanadinoethanesulfonate were as effective as taurine, while hypotaurine and α-aminoethylhydrogen sulfate were only partially effective at high concentrations, and isethionic acid was without effect. Taurine, similar to GABA enhanced the effects of pentobarbital on TBPS binding when present at concentrations that were otherwise ineffective on their own. The results of these studies support the notion that taurine interacts with the GABA recognition site of the GABA A receptor complex.

The novel antiepileptic drug levetiracetam (ucb L059) appears to act via a specific binding site in CNS membranes

Levetiracetam (( S)-α-ethyl-2-oxo-pyrrolidine acetamide, ucb L059) is a novel potential antiepileptic agent presently in clinical development with unknown mechanism of action. The finding that its anticonvulsant activity is highly stereoselective (Gower et al., 1992) led us to investigate the presence of specific binding sites for [ 3H]levetiracetam in rat central nervous system (CNS). Binding assays, performed on crude membranes, revealed the existence of a reversible, saturable and stereoselective specific binding site. Results obtained in hippocampal membranes suggest that [ 3H]levetiracetam labels a single class of binding sites ( n H = 0.92 ± 0.06) with modest affinity ( K d = 780 ± 115 nM) and with a high binding capacity ( B max = 9.1 ± 1.2 pmol/mg protein). Similar K d and B max values were obtained in other brain regions (cortex, cerebellum and striatum). ucb L060, the ( R) enantiomer of levetiracetam, displayed about 1000 times less affinity for these sites. The binding of [ 3H]levetiracetam is confined to the synaptic plasma membranes in the central nervous system since no specific binding was observed in a range of peripheral tissues including heart, kidneys, spleen, pancreas, adrenals, lungs and liver. The commonly used antiepileptic drugs carbamazepine, phenytoin, valproate, phenobarbital and clonazepam, as well as the convulsant agent t-butylbicyclophosphorothionate (TBPS), picrotoxin and bicuculline did not displace [ 3H]levetiracetam binding. However, ethosuximide (p K i = 3.5 ± 0.1), pentobarbital (p K i = 3.8 ± 0.1), pentylenetetrazole (p K i = 4.1 ± 0.1) and bemegride (p K i = 5.0 ± 0.1) competed with [ 3H]levetiracetam with p K i values comparable to active drug concentrations observed in vivo. Structurally related compounds, including piracetam and aniracetam, also displaced [ 3H]levetiracetam binding. ( S) Stereoisomer homologues of levetiracetam demonstrated a rank order of affinity for [ 3H]levetiracetam binding in correlation with their anticonvulsant activity in the audiogenic mouse test ( r 2 = 0.84, n = 12, P < 0.0001). These results support a possible role of this binding site in the anticonvulsant activity of levetiracetam and substantiate the singular pharmacological profile of this compound. This site remains however to be further characterised.

Effects of the anticonvulsant losigamone and its isomers on the GABAA receptor system.

We conducted in vitro studies to clarify the possible involvement of GABAA receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+ losigamone) and AO-294 (- losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, < or = 100 microM losigamone did not affect the specific binding of [3H]GABA, [3H]flunitrazepam, or [35S]t-butyl-bicyclophosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10(-8)-10(-5) M, stimulated 36Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10(-5) M potentiated the effect of a suboptimal concentration of exogenous GABA 10(-5) M on 36 Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS-induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating 36Cl influx in the spinal cord neurons by a GABA-sensitive mechanism and at a side distant from the GABA channel.

Stable high expression of human gamma-aminobutyric acid a receptors composed of alpha and beta subünitS

Multiple classes of pharmacological agents including benzodiazepines, cage convulsants like t-butylbicyclophosphorothionate (TBPS), barbiturates and neuroactive steroids allosterically modulate the y-aminobutyric acidA receptor-chloride ionophore complex (GRC). The function of benzodiazepines requires a GRC comprised of α, β and γ subunits, while TBPS, barbiturates and neuroactive steroids will allosterically modulate GRCs comprised of only a and β subunits. Binary αβ complexes are still hypothesized to be expressed in the mammalian brain particularly during development and could contribute to the pharmacological action of neuroactive steroids and barbiturates. In order to examine binary αβ complexes we report here the establishment of stable cell lines that express high levels of human GABA a receptors comprised of α1β1, α2β1 and a3β1 subunit combinations. The apparent potencies for allosteric modulation of [ 35S]TBPS for most naturally occurring neuroactive steroids for the binary subunit combinations was similar to that of the γ-containing subunit combinations. Also discussed is the usefulness of these cell lines for the biophysical analysis of the GABa a receptor stoichiometry.

Possible pharmacological correction ways of the cerebral hemodynamic changes in hypokinesia

We investigated the effects of 1-amino-5-bromouracil on the benzodiazepine-γ-aminobutyric acid (GABA)A receptor complex to elucidate its central action. 1-Amino-5-bromouracil neither displaced nor enhanced [3H]muscimol, [35S] t-butylbi-cyclophosphorothionate (TBPS), or [3H]dehydroepiandrosterone sulfate binding to the rat brain synaptosomal membranes. The anesthesia induced by 1-amino-5-bromouracil was potentiated by diazepam, pentobarbital, and muscimol, and was antagonized by picrotoxin but not by bicuculline. 1-Amino-5-bromouracil protected mice from picrotoxin-induced seizure and slightly ameliorated TBPS-induced seizure, but did not antagonize bicuculline-induced seizure. Diazepam antagonized both the bicuculline- and the picrotoxin-induced seizure, and pentobarbital antagonized the picrotoxin- and the TBPS-induced seizure. Our in vivo studies suggest that part of the central action of 1-amino-5-bromouracil is concerned with the benzodiazepine-GABAA receptor complex including the chloride channel.

Common modes of action of γ-butyrolactones and pentylenetetrazol on the GABA A receptor-ionophore complex

The effects of pentylenetetrazol and bicyclic γ-butyrolactones of similar stereostructures were studied on the convulsant and benzodiazepine binding sites and chloride ionophore activity of the γ-aminobutyric acid (GABA A) receptor-complex. Bicyclic γ-butyrolactones displayed millimolar IC 50 values and low stereoselectivities on [ 35S]t-butylbicyclophosphorothionate (TBPS) binding to the convulsant sites in synaptosomal membranes of rat forebrains. Ring saturation of bicyclic γ-butyrolactones decreased their IC 50 values by one order of magnitude. The IC 50 values of saturated bicyclic γ-butyrolactones and pentylenetetrazol were increased by GABA versus its antagonist R 5135 (3α-hydroxy-16-amino-5β,17-aza-androstan-11-one). A bicyclic γ-butyrolactone and pentylenetetrazol accelerated the dissociation of [ 35S]TBPS, displaced [ 3H]flumazenil binding in two phases and blocked the muscimol-elicited chloride currents in patch-clamped cortical neurones in culture in a similar manner. These similar effects on binding and ionophore function support their common modes of action on the GABA A receptor-ionophore complex.