Influence of environment on GABA receptors in muricidal rats

The influence of environment (isolation) on GABA receptor numbers ( [3H]-muscimol binding sites) and affinities was investigated in specific limbic areas known to be involved with the development of muricide. Olfactory bulbs of isolated rats were found to have identical numbers of [3H]-muscimol binding sites whether or not they were muricidal. However, in the olfactory bulbs of the aggregated animals a greater than two-fold increase was found in numbers of [3H]-muscimol binding sites in those rats that were muricidal. In the amygdala muricidal rats had a 32-34% increase in [3H]-muscimol binding sites over non-muricidal rats regardless of environment. In the septum non-muricidal rats had fewer [3H]-muscimol binding sites than muricidal rats and although the trend was evident, statistical vigor was seen only in the aggregated animals. Neither muricide nor isolation significantly influenced [3H]-muscimol binding numbers in the hypothalamus. GABA Ki values were examined in all brain regions and found to be the same in the isolated and aggregated animals whether or not they were muricidal. We concluded that environment appears to influence apparent GABA receptor numbers in the olfactory bulbs and septum whereas muricidal behavior correlates well with an increase in apparent number of GABA receptors in the amygdala. GABA receptors in the hypothalamus were not influenced by either environment or aggression.     

Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.

1. The modulation of the gamma-aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage-clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]-muscimol to a preparation of porcine brain membranes. 2. The progesterone metabolites 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one (greater than or equal to 30 nM) reversibly and dose-dependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 microM), and over the concentration range 30 nM-100 microM stimulated the binding of [3H]-muscimol. In contrast, 5 alpha- and 5 beta-pregnan-3 beta-ol-20-one (30 nM-100 microM) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3. Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]-muscimol binding sites as the principle action of the active steroid isomers. 4. GABA-evoked currents were also potentiated by androsterone (1 microM) and the deoxycorticosterone metabolite 5 alpha-pregnane-3 alpha,21-diol-20-one (100 nM). 5. Secobarbitone (10-100 microM), pentobarbitone (10-300 microM) and phenobarbitone (100-500 microM) reversibly and dose-dependently potentiated the amplitude of GABA-evoked currents in the absence of any change in their reversal potential. 6. At relatively high concentrations (greater than or equal to 30 microM) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor-channel activation since they share a common reversal potential with GABA-evoked responses (approximately 0 mV), are reversibly antagonized by bicuculline (3 microM), and potentiated by either diazepam (1 microM) or 5 beta-pregnan-3 alpha-ol-20-one (500 nM). 7. Secobarbitone (1 microM-1 mM) dose-dependently enhanced the binding of [3H]-muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8. A saturating concentration (1 mM) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5 beta-pregnan-3 alpha-ol-20-one (30 nM-100 microM). Similarly the steroid, at a concentration of 100 microM, did not influence the enhancement of [3H]-muscimol binding by secobarbitone (1 microM-1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Saturable, high affinity binding of the nucleoside transport inhibitor, nitrobenzylthioinosine, to guinea pig cardiac membranes

The site-specific binding of the potent nucleoside transport inhibitor, [3H]nitrobenzylthioinosine ([3H]NBMPR), to guinea pig cardiac membranes was rapid, reversible and saturable. [3H]NBMPR bound with high affinity to a single class of sites at which the KD was 0.23 +/- 0.07 nM and which had a Bmax of 1700 +/- 290 fmol/mg protein. Several recognized nucleoside transport inhibitors and benzodiazepines inhibited the binding of [3H]NBMPR with an order of potency similar to that observed for the inhibition of the binding of [3H]NBMPR to human erythrocytes and guinea pig synaptosomes.     

The modulation by chlormethiazole of the GABAA-receptor complex in rat brain.

1. The interactions of chlormethiazole with gamma-aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA-receptor complex and the GABAB-receptor have been studied in the rat. The GABAA-receptor was studied using [3H]-muscimol, [3H]-flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]-butyl-bicyclophosphorothionate ([35S]-TBPS) to label the chloride channel. 2. Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]-baclofen binding to the GABAB-receptor. 3. Chlormethiazole (IC50 = 140 microM) and pentobarbitone (IC50 = 95 microM) both inhibited [35S]-TBPS binding by increasing the rate of [35S]-TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]-TBPS binding. 4. Chlormethiazole enhanced the binding of [3H]-muscimol but had no effect on [3H]-flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]-muscimol and [3H]-flunitrazepam binding. 5. It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA-receptor. However, chlormethiazole does not interact with the GABAA-receptor complex in an identical manner to the sedative barbiturate pentobarbitone.     

(+)-Hydrastine, a potent competitive antagonist at mammalian GABAA receptors.

1. (+)-Hydrastine is a phthalide isoquinoline alkaloid, isolated from Corydalis stricta. It has the same 1S,9R configuration as the competitive GABAA receptor antagonist bicuculline and is the enantiomer of the commercially available (-)-hydrastine. 2. (+)-Hydrastine (CD50 0.16 mg kg-1, i.v.) was twice as potent as bicuculline (CD50 0.32 mg kg-1, i.v.) as a convulsant in mice. This action was stereoselective in that (+)-hydrastine was 180 times as potent as (-)-hydrastine. 3. (+)-Hydrastine was a selective antagonist at bicuculline-sensitive GABAA receptors in the guinea-pig isolated ileum. It did not influence phaclofen-sensitive GABAB receptors or acetylcholine receptors in this tissue. (+)-Hydrastine was a competitive antagonist of GABAA responses (pA2 6.5) more potent than bicuculline (pA2 6.1). 4. When tested against the binding of [3H]-muscimol to high affinity GABAA binding sites in rat brain membranes, (+)-hydrastine (IC50 2.37 microM) was 8 times more potent than bicuculline (IC50 19.7 microM). 5. As an antagonist of the activation of low affinity GABAA receptors as measured by the stimulation by GABA of [3H]-diazepam binding to rat brain membranes, (+)-hydrastine (IC50 0.4 microM) was more potent than bicuculline (IC50 2.3 microM). 6. (+)-Hydrastine, 10 nM to 1 mM, did not inhibit the binding of [3H]-(-)-baclofen to GABAB binding sites in rat brain membranes.     

Lack of allosteric modulation of striatal GABA(A) receptor binding and function after cocaine sensitization

GABA(A) receptor binding after repeated cocaine has been shown to be either increased as indicated by benzodiazepine binding or decreased as indicated by convulsant-site binding. We measured the GABA binding site with [3H]-muscimol binding to GABA(A) receptors and found no differences between saline- and cocaine-sensitized rats. Allosteric modulation of [3H]-muscimol binding with flunitrazepam was also unchanged after cocaine sensitization. In addition, [3H]-flunitrazepam binding and allosteric modulation of [3H]-flunitrazepam binding with GABA was unchanged after 1 day withdrawal from repeated cocaine. GABA(A) receptor function and allosteric modulation of GABA(A) receptor function measured by GABA-stimulated Cl(-) uptake was also unchanged after withdrawal from repeated cocaine. Finally, in vitro cocaine reduced GABA(A) receptor function in striatal microsacs of saline- and cocaine-treated rats. In conclusion, repeated cocaine did not change the coupling of the GABA(A) receptor between the GABA and benzodiazepine (BZD) binding site after 1 day withdrawal.     

Inhibition by arylsulphatase A of Na-independent [3H]-GABA and [3H]-muscimol binding to bovine cerebellar synaptic membranes

Over an identical concentration range, both [³H]-GABA and [³H]-muscimol were bound to bovine cerebellar synaptic membranes through a sodium-independent and high-affinity process. Preincubating the synaptic membrane suspensions with arylsulphatase A inhibited [³H]-GABA and [³H]-muscimol binding. Furthermore, by using 4-nitrocatechol sulphate as a substrate for arylsulphatase A, a direct correlation appeared to exist between the amounts of SO^2?in4 released and the degree of inhibition of [³H]-GABA binding to synaptic membranes. The inhibitory effect of arylsulphatase A was blocked by known inhibitors of arylsulphatase such as AgNO₃. Scatchard analysis of specific [³H]-GABA binding to synaptic membranes showed that arylsulphatase decreased the number of [³H]-GABA binding sites from 2.65 to 1.75 pmol mg proteins without changing appreciably the affinity of [³H]-GABA binding to cerebellar synaptic membranes. These observations are interpreted to indicate that some components of GABA recognition sites may also contain sulpholipid(s) in addition to phospholipid(s).     

Binding characteristics of [3H]quinupramine to rat brain membrane fractions

The binding characteristics of [3H]quinupramine to rat brain membrane fractions were studied. The specific binding of [3H]quinupramine to rat brain membrane fractions was stable, reversible and saturable. Scatchard analysis of the data from saturation experiments indicated that the specific binding was a single population with an affinity (KD) of 3.04 nM, a maximal binding site number (Bmax) of 714 fmol/mg protein, and a Hill coefficient (nH) of 1.08. Compounds known to inhibit muscarinic cholinergic receptors such as atropine and quinuclidinyl benzilate were the most potent competitors of [3H]quinupramine binding. When the drug potencies in inhibiting [3H]quinupramine binding were tested in the presence of 10 nM atropine, mianserin was the most potent competitor. Studies of the subcellular fractions showed that there was an enrichment of [3H]quinupramine binding sites in the synaptosome fraction. The regional distribution study revealed the highest densities of binding sites in the cerebral cortex and the lowest in the cerebellum. Thus, the specific binding of [3H]quinupramine observed here can be accounted for by both muscarinic cholinergic and serotonin S2 receptors.     

The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of propranolol with GABA stimulated diazepam binding to rat brain membranes

1. In the case of an intraperitoneal propranolol injection to rats (25 mg/kg), there was no modification in benzodiazepine and gamma-amino-butyric acid recognition sites since [3H]diazepam or [3H]-muscimol binding was not affected. 2. There was an increase in GABA stimulated-diazepam binding (100%, statistical risk lower than 1%). 3. This last result suggests that propranolol treatment can stimulate GABA-benzodiazepine receptor coupling.     

Binding of the cage convulsant, [3H]TBOB, to sites linked to the GABAA receptor complex

[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.     

GABA preincubation of rat brain sections increases [3H]GABA binding to the GABAA receptor and compromises the modulatory interactions

Receptor autoradiography has been employed to investigate the effect of gamma-aminobutyric acid (GABA) preincubation on the interaction of the GABAA receptor with its ligands. [3H]GABA (50 nM) binding to the GABAA receptors is increased by 60% compared to control sections after GABA (100 microM) preincubation. Receptor autoradiography shows that the increase is more pronounced in certain brain areas. The allosteric interactions between the GABA and benzodiazepine recognition sites were also examined. An increase in [3H]GABA (50 nM) binding to rat brain sections by co-incubation with the benzodiazepine, flunitrazepam (FNZ) has been observed autoradiographically. This effect has been quantitated in several brain regions; the overall brain increase in [3H]GABA binding induced by 1 microM FNZ was 20%. The increase in [3H]FNZ (1 nM) binding by co-incubation with GABA has also been observed autoradiographically, and the effect quantitated in four brain regions. The overall brain increase in [3H]FNZ binding induced by 100 microM GABA was 34%. After GABA preincubation these allosteric responses are significantly reduced in size. The increase in the [3H]GABAA binding as a consequence of GABA preincubation appears to reflect an increase in receptor affinity for [3H]GABA with no significant change in the maximum number of binding sites. We suggest that GABA preincubation converts the GABAA receptor to a higher affinity desensitised receptor conformation.     

The effects of chronic haloperidol administration on GABA receptor binding

The effect of chronic administration of haloperidol for 30 days and its subsequent withdrawal for 0,4 and 8 days on the binding of [³H]-GABA and [³H]-muscimol to GABA binding sites in rat brain membranes obtained from six discrete regions of the brain and spinal cord was investigated. The chronic administration of haloperidol resulted in a marked increase in the number of GABA binding sites within the substantia nigra, but did not affect GABA binding sites in other regions of rat brain or spinal cord. The increase in GABA binding sites in the substantia nigra was evident for at least 4 days following termination of haloperidol treatment; however, 8 days following withdrawal from haloperidol, when dopaminergic supersensitivity had become maximally expressed, the number of GABA binding sites in the substantia nigra had declined and was the same as that observed in the control rats. These observations suggest that blockade of dopamine receptors in the striatum by haloperidol results in a compensatory decrease in the activity of the GABAergic strionigral system that leads to an increase in the number of GABA binding sites within the substantia nigra. This increase in the number of GABA binding sites is gradually reversed when dopamine is again allowed to interact with its receptors.     

Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 membrane

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation produced and opioid induced analgesia. In the present study the opioid binding characteristics of the PAG were examined with an in vitro radioligand binding technique. [3H]Ethylketocyclazocine (EKC), 2 nM, was used as a tracer ligand to nonselectively label mu, delta, and kappa binding sites in PAG enriched P2 membrane. The mu selective ligand [D-Ala2,N-methylPhe4,Glyol5]enkephalin (DAGO) competed with [3H]EKC for more than one population of binding sites with both high and low affinity. In contrast the delta selective ligand [D-Pen2,D-Pen5]enkephalin (DPDPE) and the kappa selective ligand trans-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, methane sulfonate, hydrate (U50,488H) each competed with [3H]EKC for a single population of binding sites with low affinity. DPDPE and U50,488H also competed with 2 nM [3H]DAGO for a single population of binding sites with similar low affinity. DAGO and not DPDPE competed with 2 nM [3H][D-Ala2,D-Leu5]enkephalin (DADLE) with high affinity. 2 nM [3H]DPDPE did not substantially label PAG enriched P2 membrane, and 1 nM DAGO competed with all specific [3H]DPDPE binding which was observed. These binding data are consistent with the presence of a single population of mu selective high affinity binding sites in PAG enriched P2 membrane to which delta ligands and kappa ligands have low affinity.     

[3H]-lifarizine,a high affinity probe for inactivated sodium channels.

1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528) on benzodiazepine receptor bindings and gamma-aminobutyric acid release

The action of a new type of anti-anxiety compound, 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528), was tested on benzodiazepine receptor bindings and on [3H]-GABA release. BP-528 did not alter [3H]-diazepam binding to rat cerebral cortical and hippocampal membranes either in the presence or absence of GABA; and the binding of [3H]-propyl-beta-carboline-3-carboxylate at low concentration (0.04 nM), which labels only the type I benzodiazepine receptor, was not changed by BP-528. BP-528 did not interact with the GABA-benzodiazepine receptor complex, which is related to the anti-anxiety activity of benzodiazepines. This compound affected neither GABA binding nor GABA uptake. Ten micromolar BP-528 depressed high K+-induced [3H]-GABA release from preloaded rat hippocampal slices. However, the same concentration of BP-528 also inhibited high K+-induced calcium uptake by rat cerebral cortical synaptosomes.     

gamma-Aminobutyric acid release from synaptosomes as influenced by Ca2+ and Ca2+ channel blockers

We studied the correlation between the high affinity binding of Ca2+ channel blockers to purified synaptic plasma membranes (SPM) and the effect of these drugs in blocking the 45Ca2+ uptake and the release of [3H]gamma-aminobutyric acid [( 3H]GABA) by preloaded synaptosomes. The Ca2+ channel blocker binding sites were characterized by studying the binding of the dihydropyridine, [3H]nimodipine, and of the phenylalkylamine, (-)-[3H]desmethoxyverapamil, to purified SPM isolated from sheep brain cortex synaptosomes. The purified SPM had high affinity binding sites for both Ca2+ channel blockers. The binding parameters were similar to those previously reported for whole brain homogenates: KD = 0.64 nM and Bmax = 160 fmol/mg of protein for [3H]nimodipine, and KD = 7.9 nM and Bmax = 1,500 fmol/mg of protein for (-)-[3H]desmethoxyverapamil. The Ca2+ channel blockers inhibited the release of [3H]GABA induced by K+ depolarization in the presence or in the absence of Ca2+. The Ca2+-dependent component of [3H]GABA release was inhibited by verapamil, (-)-D 600, d-cis-diltiazem, nifedipine and PY 108-86 with IC50 values of 2.2 X 10(-5) M, 6.3 X 10(-5) M, 3 X 10(-4) M, greater than 10(-4) M and 3 X 10(-5) M, respectively. Furthermore, the Ca2+ channel blockers also inhibited the Ca2+-independent [3H]GABA release which occurred in the presence, but not in the absence, of external Na+. The Ca2+ channel blockers at concentrations which inhibited [3H]GABA release inhibited the entry of Ca2+ through the Ca2+ channels and also the entry of Ca2+ by Na+/Ca2+ exchange. We conclude that the concentrations of Ca2+ blockers necessary to block Ca2+ uptake through the Ca2+ channels and by Na+/Ca2+ exchange coincide with the concentrations at which they inhibit [3H]GABA release, but that their effect on the relationship between Ca2+ uptake and [3H]GABA release is different for the various blockers. The effects of the drugs on Ca2+ movements and [3H]GABA release are not specifically mediated through the high affinity binding of the drugs since relatively high concentrations were necessary (greater than 10(-5) M) for the effects reported here.     

Further studies on the biochemical characterization and autoradiographic distribution of [H]hemicholinium-3 binding sites in rat brain: a presynaptic cholinergic marker

Hemicholinium-3 (HC-3) is a potent inhibitor of the high-affinity choline uptake system (HACU). Here we report on the biochemical characterization and autoradiographic distribution of [3H]hemicholinium-3 binding sites in rat brain, confirming and expanding results from previous studies. The binding of [3H]HC-3 to striatal membranes was specific, to a single site, sodium-dependent, saturable, and of high-affinity, Kd values being about 3 nM for striatum, 5 nM for the hippocampus and 12 nM for neocortex. [3H]HC-3 specific binding exhibited a pharmacological profile suggestive of physiologically relevant interactions and fully comparable to that reported for HACU. The uneven distribution of [3H]HC-3 binding sites exhibited a high degree of correspondence with the reported distribution of HACU and other enzymatic presynpatic cholinergic markers. The punctual differences between our study and previous works on [3H]HC-3 binding are analysed. We conclude that [3H]HC-3 labelling may be used as a selective and quantifiable marker of the cholinergic presynaptic terminals in close relationship with HACU.     

Action of pyrazolopyridines as modulators of [3H]flunitrazepam binding to the gaba/benzodiazepine receptor complex of the cerebellum

The pyrazolopyridines etazolate (SQ 20009) and cartazolate (SQ 65396) have strong modulatory effects on the GABA/benzodiazepine receptor complex of rate cerebellum. Thus, etazolate and cartazolate directly stimulate [3H]flunitrazepam binding (with EC50 values of 1.2 microM and 0.3 microM respectively) by increasing the apparent affinity of [3H]flunitrazepam for its binding sites. Stimulation of [3H]flunitrazepam binding by pyrazolopyridines is dependent on the presence of certain anions like chloride, bromide, iodide, nitrite, nitrate but not fluoride, acetate, formate or sulfate. If is inhibited by bicuculline-methiodide, and by the "chloride channel drugs' picrotoxinin and IPTBO. isoTHAZ, a GABA analogue with GABA antagonist properties in vivo, fails to inhibit binding stimulated by etazolate but antagonizes [3H]flunitrazepam binding stimulated by GABA. The pyrazolopyridines have also indirect effects on benzodiazepine receptor binding since they enhance the apparent sensitivity of those GABA recognition sites which are coupled to benzodiazepine binding sites. Thus, in the presence of 10 microM etazolate, GABA and muscimol enhance [3H]flunitrazepam binding, with EC50 values of 109 nM and 12 nM respectively. This sensitization effect is partially dependent on the presence of chloride ions. The pyrazolopyridines facilitate also the stimulation of benzodiazepine receptor binding by beta-alanine and taurine and by the rigid and flattened GABA analogues THIP and piperidine-4-sulfonic acid. Taken together, these results suggest that the pyrazolopyridines modulate [3H]flunitrazepam binding by acting at a site closely related to GABA receptor-regulated chloride ion channels.     

Regional mirex distribution and its effects on gamma-aminobutyric acid and flunitrazepam binding in mouse brains

Following ip injection of [14C]mirex, its regional distribution was studied parallel to its in vitro effects on [3H]-gamma-aminobutyric acid (GABA) and [3H]flunitrazepam (FNZ) binding to, and [3H]GABA release from, synaptosomes of various mouse brain regions, in order to determine the relationship between relative mirex distribution and its neurotoxic effects mediated through the GABA receptor-ionophore complex. The pattern of mirex uptake into cerebral cortex (CC), brainstem (BS), and cerebellum (CB) showed an initial linear dose-dependent uptake, followed by a decline at higher concentration. The Vmax and Km values determined for the linear mirex uptake phase indicated varied affinities by brain regions, CB and BS being more susceptible to mirex uptake than CC. Both synaptosomal GABA binding and FNZ binding were significantly reduced by mirex in the order of BS approximately equal to CB greater than CC for GABA, and BS approximately equal to CC greater than CB for FNZ. However, mirex lacked any significant effect on the Ca2+-dependent, K+-stimulated release of GABA from radio-prelabeled synaptosomes. While the data indicate no significant differences between brain regions in mirex uptake, they suggest that regional specificities do exist with respect to the inhibition caused by mirex on GABA and FNZ binding to synaptosomes. Unlike the major effects of chlordecone (an analog of mirex) on the dopaminergic system, mirex seems to be primarily neurotoxic through its more specific interaction with the GABA and FNZ binding sites.