Dissociation of [35S]t-Butylbicyclophosphorothionate Binding Differentiates Convulsant and Depressant Drugs that Modulate GABAergic Transmission

The dissociation of [35S]t-butylbicyclophosphorothionate ([35S]TBPT) from binding sites on membranes from rat cerebral cortex, after addition of saturating concentrations of convulsant and depressant drugs, was studied. The addition of unlabeled TBPT, picrotoxinin, or pentamethylenetetrazol resulted in dissociation patterns that were monophasic and not distinguishable, suggesting that these convulsants bind competitively to the same (convulsant) sites. In contrast, gamma-aminobutyric acid (GABA) greatly facilitated [35S]TBPT dissociation by binding allosterically to the GABA recognition site of the receptor-ionophore complex. TBPT dissociation was similarly accelerated by the depressants etazolate, (+)-etomidate, and barbiturates. The convulsant and depressant S(+) and R(-) stereoisomers of N-methyl-5-phenyl-5-propyl-barbituric acid displayed large stereoselectivity in the acceleration of TBPT dissociation. These results suggest that depressants bind to sites different from the convulsant sites of the allosteric GABA receptor complex, or the binding of depressants to the same population of sites elicits negative cooperativity and dissociates the convulsants.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Heterotropic Cooperativity Between Putative Recognition Sites for Progesterone Metabolites and the Atypical Benzodiazepine Ro 5–4864

The binding of the cage convulsant t-butylbicyclophosphorothionate (TBPS) and 36Cl- uptake by synaptoneurosomes were used to test the ability of progesterone metabolites to modulate allosterically the Ro 5-4864 (4'-chlorodiazepam) binding site that is functionally coupled to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex (GBRC) in rat brain. Dose-dependent enhancement of [35S]TBPS binding by Ro 5-4864 occurs in rat cerebral cortex in the presence of the progesterone metabolites 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha, 20 alpha-diol (pregnanediol). The pregnanediol effect is completely GABA dependent, whereas that of 3 alpha-OH-DHP is not. Conversely, Ro 5-4864 opposed the action of 3 alpha-OH-DHP by increasing the IC50 for 3 alpha-OH-DHP inhibition of [35S]TBPS binding. In cortical synaptoneurosomes, Ro 5-4864 antagonized both 3 alpha-OH-DHP and pregnanediol enhancement of GABA-stimulated 36Cl- uptake. In both binding and functional studies, pregnanediol showed limited efficacy relative to 3 alpha-OH-DHP, as previously reported. These findings provide the initial evidence that the GBRC-linked Ro 5-4864 binding site is allosterically coupled to the putative progesterone metabolite recognition site and confirm the GABA-mimetic properties of 3 alpha-OH-DHP and pregnanediol.     

Histidine modification with diethylpyrocarbonate induces a decrease in the binding of an antagonist, PK 11195, but not of an agonist, RO5-4864, of the peripheral benzodiazepine receptors

[3H]PK 11195 binding to peripheral type benzodiazepine binding sites in kidney membranes is inhibited by the histidine blocking agent diethylpyrocarbonate. This reagent irreversibly decreases the Bmax for [3H]PK 11195 without affecting the affinity. By contrast binding of [3H]RO5-4864 is not affected by diethylpyrocarbonate treatment. However RO5-4864 can protect in a concentration dependent manner the [3H]PK 11195 binding site from diethylpyrocarbonate whereas clonazepam and RO15-1788 are not active. These results suggest that PK 11195 and RO5-4864 interact with different conformational states of the receptors that RO5-4864. This is in agreement with our previous hypothesis that PK 11195 is an antagonist and RO5-4864 an agonist at the "peripheral type" benzodiazepine receptors.     

Convulsant/depressant site of action at the allosteric benzodiazepine-GABA receptor-ionophore complex

Several classes of centrally acting convulsant, depressant, anticonvulsant and anxiolytic drugs modulate GABAergic transmission. The postsynaptic receptor with which these drugs interact is an allosteric complex with distinct binding sites for GABA, benzodiazepines, picrotoxinin and related compounds. Convulsants which inhibit GABA transmission (except bicuculline) inhibit competitively the binding of dihydropicrotoxinin (DHP) or t-butylbicyclophosphorothionate (TBPT) to the picrotoxinin site and prevent the allosteric enhancing effect of depressant drugs on GABA and benzodiazepine binding. Depressant drugs give a mixed inhibition of TBPT binding. The possible topography of the picrotoxinin site and its relationship to convulsant/depressant drug action at the benzodiazepine-GABA receptor-ionophore complex is discussed.     

Characterization of the Binding of [3H]Ro 5-4864, a Convulsant Benzodiazepine, to Guinea Pig Brain

The density of high affinity binding sites for [3H]4'-chlorodiazepam [( 3H]Ro 5-4864) in guinea pig cerebral cortex is significantly higher (3.8-fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5-4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5-4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.     

Hormonal interactions with benzodiazepine binding sites in vitro

Prostaglandin A1 and hormones like corticosteroids and DL-Thyroxin (T4) inhibit binding of [3H]RO 5-4864 and [3H] Clonazepam to their respective binding sites with inhibition constants in the low micromolar range. The corticosteroid Cortisone inhibits [3H] RO 5-4864, but not [3H] Clonazepam binding in a competitive manner with an inhibition constant of 4.3 +/- 0.7 microM, Prostaglandin A1 inhibits [3H] Clonazepam, but not [3H] RO 5-4864 binding in a competitive manner with an inhibition constant of 6 +/- 1.2 microM and DL-Thyroxin (T4) inhibits both [3H] RO 5-4864 and [3H] Clonazepam binding with inhibition constants of 12.1 +/- 2.2 and 1.6 +/- 0.4 microM respectively. While the inhibition of [3H] RO 5-4864 binding by DL-Thyroxin (T4) is competitive, the inhibition of [3H] Clonazepam binding is of the mixed type as indicated by Scatchard Plot.     

"Peripheral-Type"Binding Sites for Benzodiazepines in Brain: Relationship to the Convulsant Actions of Ro 5–4864

Previous studies have shown that Ro 5-4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5-4864 were not mediated by "brain-type" benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5-4864 for their capacities to displace [3H]Ro 5-4864 from "peripheral-type" binding sites and their potencies as convulsants (or as antagonists of Ro 5-4864-induced convulsions). It was observed that compounds such as KW 3600 (the N-desmethyl analog of Ro 5-4864), which have very low affinities for "peripheral-type" sites, are convulsants with a potency nearly equal to that of Ro 5-4864. In contrast, compounds such as Ro 5-6900 and PK 11195, which bind with very high affinities to "peripheral-type" binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5-4864. Within a series of compounds that are structurally related to Ro 5-4864 there is a good correlation (r = 0.93; p less than 0.01) between their potencies as convulsants and their capacities to displace [35S]t-butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of "peripheral-type" binding sites by high-affinity ligands may not be directly involved in the convulsant actions of Ro 5-4864 and related compounds.     

Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3 isoquinolinecarboxamide. II. In vivo studies

Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.     

Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain

A series of tetrazole convulsants were examined for their potencies in displacing [35S]-t-butylbicyclophosphorothionate (TBPS) from the picrotoxin site on the benzodiazepine-GABA-chloride ionophore receptor complex. All of the tetrazole derivatives tested inhibited [35S]-TBPS binding from rat forebrain membranes, and except for one (undecamethylenetetrazole), had Hill coefficients near unity. Similar to other chemically unrelated convulsants the inhibition of [35S]-TBPS binding by the various tetrazole derivatives was unaffected by the addition of the bicucculine-like GABA antagonist, R 5135. To ascertain whether the inhibition of specific [35S]-TBPS binding by the tetrazole derivatives was related to their convulsant properties, we compared their in vitro potencies in displacing [35S]-TBPS binding with their minimum convulsant potencies in mice. A very good correlation was observed (r = 0.96, p less than 0.001) between their relative affinities for the [35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produce their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Clozapine and Some Other Antipsychotic Drugs May Preferentially Block the Same Subset of GABAA Receptors

Selective blockade of a subset of GABA_A receptors may be involved in the antipsychotic effects of Clozapine and several other antipsychotic drugs. Seven antipsychotic drugs, and 11 drugs classified as antidepressants that only partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, do not yield additive reversal when tested pairwise with Clozapine, which also only partially reverses the inhibitory effect of GABA. This suggests that all of these antipsychotic/antidepressant drugs may block a common subset of GABA_A receptors. DMCM and Ro 5-4864 are also partial reversers of GABA's inhibitory effect, but they yield additive reversals when tested pairwise with the antipsychotic/antidepressant drugs, and also with each other, suggesting that DMCM, Ro 5-4864, and the antipsychotic drugs define three heterogeneous subsets of GABA_A receptors, with variable overlap, depending on the drug. Several potent ligands for benzodiazepine binding sites can block the GABA inhibitory effects of DMCM and Ro 5-4864, but with different patterns: the ligands generally blocked DMCM less potently, but more completely than Ro 5-4864, Ro 5-4864 was not blocked by Flumazenil or CGS-8216, ligands that potently blocked DMCM. Nine additional antipsychotic/antidepressant drugs, as well as Clozapine, and 7 classical GABA_A receptor blockers, all of which reversed GABA nearly completely, when tested at lower concentrations that only reverse 20–35%, yielded almost complete additivity when tested pairwise with DMCM or Ro 5-4864. Another convulsant benzodiazepine, KW-1937, a positional isomer of Brotizolam, fully reverses the inhibitory effect of 1 M GABA. At a lower concentration yielding about 50% reversal, KW-1937 is completely additive with DMCM, but entirely nonadditive with Ro 5-4864. The 50% reversal obtained with KW-1937 was potently blocked by Triazolam, but with a plateau similar to that obtained with Ro 5-4864. The results with KW-1937 suggest that its 50% reversal largely corresponds to the reversal obtained with Ro 5-4864, and that virtually all of the [³⁵S]TBPS binding sites inhibited by 1 M GABA are coupled to benzodiazepine binding sites. The fraction of GABA_A receptors preferentially blocked by all the antipsychotic/antidepressant drugs, roughly 25% of the [³⁵S]TBPS binding sites inhibited with 1 M GABA, are sensitive to KW-1937, but not to DMCM or to Ro 5-4864.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies

The [3H]PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide, binding sites in rat cardiac membranes are saturable, with high affinity, specific GABA-independent and correspond to the peripheral type of benzodiazepine. The order of potency of displacing agents was: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. The Bmax obtained with [3H]PK 11195 was equivalent of the Bmax obtained with [3H]RO5-4864 in the same experimental conditions. However thermodynamic analysis indicates that the [3H]PK 11195 binding was entropy driven whereas the [3H]RO5-4864 binding was enthalpy driven. Consequently PK 11195 might be an antagonist of these binding sites and RO5-4864 an agonist or a partial agonist. The simultaneous use of both drugs might help to elucidate the physiological relevance of peripheral benzodiazepine binding sites.     

Selective pharmacological modulation of renal peripheral-type benzodiazepine binding by treatment with diuretic drugs

We have assessed the effects of in vivo administration of different classes of diuretic drugs on the expression of the peripheral-type benzodiazepine binding site (PBBS) in crude membranes derived from the cortex and outer medulla of rat kidney by saturation analysis with the PBBS-selective ligands [3H]RO5-4864 and [3H]PK 11195 in cortex and [3H]RO5-4864 in outer medulla. Administration for 14-15 days of furosemide, a drug that blocks NaCl-KCl coupled transport in the thick ascending limb of the loop of Henle, produced a significant doubling in the PBBS density (Bmax) in outer medulla, a region of the kidney rich in thick ascending limbs, and produced a lesser but significant increase in PBBS density in the cortex. Conversely, administration for 14-15 days of the carbonic anhydrase inhibitor acetazolamide, which acts predominantly in the proximal tubule, and hydrochlorothiazide, which acts predominantly in the early distal tubule, elicited statistically significant increases in PBBS density in renal cortex but not in renal outer medulla. Furthermore, all drug treatments were without effect on the equilibrium dissociation constants (Kds) of [3H]RO5-4864 and [3H]PK 11195 binding to cortical and outer medullary membrane preparations. These findings demonstrate that the PBBS can be selectively "up-regulated" in different regions of the kidney by diuretic drugs with different modes/sites of action.     

Labelling of "Peripheral-Type" Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] is a new ligand for the "peripheral-type" benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50 congruent to 10(-9) M) [3H]-RO5-4864 (a benzodiazepine which specifically labels the peripheral-type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD = 10(-9) M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral-type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5-4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.     

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.     

The Peripheral-Type Benzodiazepine Receptor Is Present in Astrocytes but Is Not a Primary Site of Action for Convulsants/Anticonvulsants

Abstract: High-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of both mouse neocortical and cerebellar astrocytes. The binding sites were enriched in mitochondrial fractions on differential centrifugation. An 18-kDa polypeptide was specifically photolabelled in cerebellar astrocytes by [³H]-PK 14105, a photolabel for the peripheral-type benzodiazepine receptor. However, this polypeptide did not show any reactivity with an antiserum previously raised against the corresponding polypeptide from rat adrenal gland. Various anticonvulsant and convulsant agents were tested for their ability and potency at inhibiting [³H]Ro 5-4864 binding to neocortical astrocytes. Many of these compounds, previously reported to be inhibitors of diazepam binding to neocortical astrocytes, proved ineffective in this study. No correlation was observed between convulsant/anticonvulsant potency and ability to inhibit [³H]Ro 5-4864 binding to the peripheral-type benzodiazepine receptor in these cells. Thus, whereas some convulsants and anticonvulsants might interact with this astrocytic receptor, such a system has no validity as a general screening method for these agents.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

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.