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Within the context of the discussion about rational polytherapy, we determined the effects of four anaesthetics on the binding of [3H]t-butylbicycloorthobenzoate ([3H]TBOB) to the GABA(A) receptor complex in the presence of several concentrations of GABA (gamma-aminobutyric acid), in order to build a molecular model that can describe and quantify the interactions between the compounds. The empirical isobole method revealed that GABA and the anaesthetics acted synergically in displacing [3H]TBOB. This synergy could be described by a simple molecular model in which both GABA and the anaesthetics displaced [3H]TBOB allosterically and in which GABA allosterically enhanced the binding of the anaesthetics. To get information about the interaction between GABA and anaesthetics, we used [3H]TBOB as a tracer ligand. The model indicated that GABA enhanced the affinity of thiopental 3.0-fold, propofol 5.0-fold, the neuroactive steroids Org 20599 3.5-fold and Org 20549 13-fold. Insight into the molecular mechanism and strength of these interactions can help clinicians to choose therapeutically optimal drug and dose combinations: a step towards rational polytherapy.

[3H]-t-Butylbicycloorthobenzoate ([3H]TBOB), a convulsant, is known to label a binding site on the GABAA receptor complex. Bicuculline methochloride (bicuculline MCl), folic acid, pentazocine, naloxone, ethyl-beta-carboline-3-carboxylate (beta CCE) and Ro 5-4864 have (pro)convulsive properties in vivo. In the present study, we determined the extent to which these compounds modify the binding of [3H]TBOB in the presence of IC50 amounts of GABA (5 microM) or diazepam (50 microM). We found that the GABA antagonist bicuculline MCl reversed the inhibitory effect of GABA on [3H]TBOB binding completely, as was expected. Folic acid, pentazocine and naloxone also reversed the inhibitory effect of GABA on [3H]TBOB binding. This finding is compatible with the view that the proconvulsive effects of these compounds can be credited to a reduction of GABAergic action at the GABAA receptor complex. We suggest that the reversal of GABA's inhibition of [3H]TBOB binding is a sufficient (but not a necessary) condition to predict proconvulsive (side) effects of drugs. beta CCE and Ro 5-4864 modified [3H]TBOB binding in the presence of GABA in a biphasic fashion. A unique relation between beta CCE, Ro 5-4864 and the GABAA complex might exist. Bicuculline MCl reversed the inhibitory effect of diazepam on [3H]TBOB binding only partly. beta CCE did not reverse the inhibitory effect of diazepam on [3H]TBOB binding, neither did Ro 5-4864. The presence of a GABA-independent interaction between a low affinity benzodiazepine recognition site and the TBOB site is proposed.

[3H]t-Butylbicycloorthobenzoate ([3H]TBOB) binds to specific sites on crude synaptic rat brain membranes. The dissociation constant, Kd, determined from saturation experiments is near 8 nM and the receptor density Bmax is about 20 pmol/g wet tissue. Non-specific binding constitutes about 35% of the total binding at 4 nM [3H]TBOB. The association of [3H]TBOB is monophasic but its dissociation is biphasic. Kd values of 8 nM (70% of the binding sites) and 20 nM (30% of the binding sites) were estimated from the kinetic data. These values differ from those previously reported. Specifically bound [3H]TBOB is displaced by picrotoxin and by t-butylbicyclophosphorothionate (TBPS). No simple competitive interaction of picrotoxin with [3H]TBOB binding was found. Micromolar quantities of the GABAergic facilitating compounds, GABA, muscimol and diazepam inhibited [3H]TBOB binding in an allosteric manner.

The biochemical mechanism responsible for the convulsive effects of folates was investigated. The epileptogenic effects of folates were determined in vivo by quantification of the seizures following intracortical application in rats. The rank order of epileptogenic effects is: folic acid greater than or equal to 5-HCO-H4 folate greater than H2 folate greater than 5-CH3-H4 folate. This sequence of epileptogenicity in vivo is compared to the rank order of the effects of folates on radioligand binding to the GABAA-receptor complex in vitro. The inhibitory potencies of folates on [3H]muscimol and [3H]diazepam bindings did not correlate with their epileptogenic effects. However, folates reverse the inhibiting effect of GABA on the binding of the cage convulsant [3H]TBOB [( 3H]t-butylbicycloorthobenzoate). The rank order of this in vitro effect (folic acid greater than 5-HCO-H4 folate greater than H2 folate = 5-CH3-H4 folate) resembles the rank order of epileptogenicity determined in vivo. A relationship between the in vivo and in vitro effects is therefore suggested.

The site of action responsible for the convulsive effect of folic acid was investigated in vitro. Folic acid (ECmax 5 x 10(-4) M) enhances the binding of the cage convulsant [3H]t-butylbicycloorthobenzoate ([3H]TBOB) to rat brain membranes, namely to 130% of control in the absence of GABA and to over 300% of control in the presence of physiological concentrations of GABA. Analysis of the binding parameters reveals that folic acid increases the apparent number of [3H]TBOB binding sites.