T-butylbicyclophosphorothionate Binding Research Articles

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors.

While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified. This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors. Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific. CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

MODULATION OF THE GABA<sub>A</sub> RECEPTOR BY SRIF IN RAT THALAMUS

Somatostatin has been reported to modulate GABAA receptor complex in many brain structures. This study was conducted to investigate somatostatin modulation of the GABAA receptor binding in several rat diencephalic structures, focusing primarily on the thalamus, as this structure plays important roles. Animals were assigned to control conditions. Changes in specific binding of GABAA receptor as labelled with [35S]t-Butylbicyclophosphorothionate (TBPS) were assessed by in vitro quantitative autoradiography with the aid of a computer assisted image analysis system. Our results reveal the presence of higher densities in several thalamic structures located principally in the part of thalamus. We demonstrate for the first time the presence of a modulatory effect of somatostatin on the GABAA receptor complex in this brain region in rats. Indeed, the peptide affected in a concentration-dependent manner; the binding of [35S]-Tertiary Butylbicyclophosphorothionate (TBPS) to the convulsant site of the GABAA receptor complex in thalamic structures with an affinity in the micromolar range (10-3 to 3.10-6M). The inhibitory effect of somatostatin is observed in all thalamic structures analyzed. The absence of a specific region effect of somatostatin on the binding of [35S]-TBPS, suggests the presence of a homogenous subunit GABAA receptor composition. Furthermore, GABA and muscimol, a GABAA receptor agonist, enhanced the affinity of somatostatin effect on [35S]-TBPS. This suggests that somatostatin allosterically modifies [35S]-TBPS binding through a mechanism similar to that of GABA.

Influences on blockade by t‐butylbicyclo‐phosphoro‐thionate of GABAA receptor spontaneous gating, agonist activation and desensitization

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1β2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1β2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)β2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1β2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)β2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1β2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1β2γ2 receptors seen with GABA (1 μm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)β2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.

Normalization of GABAA receptor specific binding in the substantia nigra reticulata and the prevention of L‐dopa‐induced dyskinesias in MPTP parkinsonian monkeys

L-Dopa therapy in Parkinson's disease (PD) is counfounded by the development of involuntary movements such as L-Dopa-induced dyskinesias (LIDs). In this study GABA(A) receptor autoradiography was assessed using [(3)H]flunitrazepam binding to the benzodiazepine site of the GABA(A) receptor and [(35)S]t-butylbicyclophosphorothionate (TBPS) binding to the chloride channel of GABA(A) receptors in the substantia nigra reticulata (SNr) and subthalamic nucleus (STN). L-Dopa-treated parkinsonian monkeys experiencing LIDs were compared to animals in which LIDs was prevented by adjunct treatments with CI-1041, a selective antagonist of the NR1A/2B subtype of NMDA receptor, or low doses of the dopamine D2 receptor agonist, cabergoline. Our results demonstrated a decrease of GABA(A) receptor specific binding in the posterior part of the SNr in dyskinetic monkeys compared to nondyskinetic animals, while no modulation has been observed in the STN. These results provide evidence for the first time that pharmacological treatments preventing LIDs in nonhuman primate model of PD are associated with normalization of GABA(A) receptor-mediated signalling in the SNr.

Effect of ocular hypertension on retinal GABAergic activity

Glutamate and γ-aminobutyric acid (GABA) are major excitatory and inhibitory retinal neurotransmitters. The balance between these signals is a key principle of organization at retinal level. Although glutamate-induced excitotoxicity could mediate retinal ganglion cell death in glaucoma, the GABAergic system was not previously examined in this disease. The aim of this work was to study the retinal GABAergic activity in eyes with ocular hypertension induced by hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 weeks of treatment with HA, GABA turnover rate, glutamic acid decarboxylase activity, and both glutamate- and high K +-induced GABA release significantly decreased, whereas GABA uptake increased in HA-treated eyes. The binding of t-butylbicyclophosphorothionate (TBPS) to GABA A/benzodiazepine Cl − channels significantly increased in eyes injected with HA as compared with vehicle-injected eyes. Changes in GABA uptake and TBPS binding persisted at 6 weeks of treatment with HA. These results indicate a dysfunction of the retinal GABAergic activity in hypertensive eyes, which could suggest the involvement of GABA in glaucomatous neuropathy.

Reduced inhibition and sensitivity to neurosteroids in hippocampus of mice lacking the GABA(A) receptor delta subunit.

The delta subunit of the gamma-aminobutyric acid (A) receptor (GABA(A)R) is expressed postnatally mostly in the cerebellum, thalamus, and dentate gyrus. Previous studies in mice with a targeted disruption of the delta subunit revealed a considerable attenuation of behavioral responses to neuroactive steroids but not to other neuromodulatory drugs. Here we show that delta subunit loss leads to a concomitant reduction in hippocampal alpha4 subunit levels. These changes were accompanied by faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate granule neurons of -/- mutants (decay tau = 25 ms) compared with +/+ controls (tau = 50 ms). Furthermore, the GABA(A)R-mediated miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in delta-mutants (tau = 6.3 ms) than controls (tau = 7.2 ms) and had decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1-10 microM), was smaller in delta-mutants (at 10 microM, 65% increase) compared with +/+ littermates (308% increase). In competition binding experiments, alphaxalone (0.03-1 microM) modulation of [35S]t-butylbicyclophosphorothionate binding was reduced in delta-mutant brain homogenates, indicating that the decreased alphaxalone effects on mIPSCs were due to changes in the GABA(A)R protein. Faster decay of evoked IPSPs and mIPSCs in delta-mutants suggests presence of the delta subunit at both synaptic and extrasynaptic GABA(A)Rs. Decreased synaptic and extrasynaptic inhibition likely contributes to the pro-epileptic phenotype of delta-mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. While the simplest explanation is that delta subunit-containing GABA(A)Rs represent the actual target of neurosteroids, it is possible that the behavioral and physiological sensitivity to neuroactive steroids is indirectly altered in the delta -/- mice.

Neuroactive steroids differ in potency but not in intrinsic efficacy at the GABA(A) receptor in vivo.

The objective of the present investigation was to characterize the in vivo EEG effects of (synthetic) neuroactive steroids on the basis of a recently proposed mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model. After intravenous administration, the time course of the EEG effect of pregnanolone, 2beta-3alpha-5alpha-3-hydroxy-2-(2,2-dimethylmorpholin-4-yl)-pregnan-11,20-dione (ORG 21465), 2beta-3alpha-5alpha-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one (ORG 20599), and alphaxalone was determined in conjunction with plasma concentrations in rats. For each neuroactive steroid the PK/PD correlation was described on the basis of a two-compartment pharmacokinetic model with an effect compartment to account for hysteresis. The observed concentration EEG effect relationships were biphasic and characterized with a mechanism-based pharmacodynamic model, which is based on a separation between the receptor activation process and the stimulus-response relationship. A single unique biphasic stimulus-response relationship could be identified for all neuroactive steroids, which was successfully described by a parabolic function. The receptor activation process was described by a hyperbolic function. Estimates for the maximum activation (e(PD)) were similar for the different neuroactive steroids but values of the potency estimate (K(PD)) ranged from 157 +/- 16 ng. ml(-1) for pregnanolone, 221 +/- 83 ng. ml(-1) for ORG 20599, and 483 +/- 42 ng. ml(-1) for alphaxalone to 1619 +/- 208 ng. ml(-1) for ORG 21465. A statistically significant correlation was observed between the in vivo potency and the IC(50) in an in vitro [(35)S]t-butylbicyclophosphorothionate binding assay (r = 0.91). It is concluded that the new PK/PD model constitutes a new mechanism-based approach to the quantification of the effects of (synthetic) neuroactive steroids in vivo effects. The results show that the neuroactive steroids differ in potency but not in intrinsic efficacy at the GABA(A) receptor in vivo.

Distribution of Inhaled m‐Xylene in Rat Brain and its Effect on GAB AA Receptor Binding

Distribution of Inhaled m‐Xylene in Rat Brain and its Effect on GAB Aa Receptor Binding: Takehiko Ito, et al. Faculty of Education, Okayama University—Organic solvents generally depress the central nervous system (CNS), similarly to volatile anesthetics. The precise mechanism of their action on the CNS, however, is not fully understood, and remains to be clarified. This study is focused on how inhaled m‐xylene distributes in the brain, and whether region specific change in GAB Aa receptor binding takes place due to the exposure. To conduct this study, we first developed a simple exposure system suitable for inhalation experiments with small animals. Using this system, six‐week‐old male Sprague‐Dawley rats were exposed to m‐xylene vapor (2000 ppm) 4 h/d, for 5 consecutive days. At the end of the exposure, m‐xylene levels in four different regions of the brain were measured by head‐space gas chromatography. Also 14μm‐thick frozen sections of the brain were made, and [35S] t‐butylbicyclophosphorothionate (TBPS) binding autoradiography was performed. Uneven distribution of m‐xylene in the brain was observed. The concentration in the cerebellum (976 ± 93.4μg/g tissue) was the highest, while that in the cerebral cortex (467 ± 43.6μ/g tissue) was the lowest. [35S]TBPS binding was significantly greater in the molecular layer of the cerebellum (control: 12.6 ± 0.64, m‐xylene 16.0 ± 1.34 fmol/mg tissue). These results suggest that m‐xylene is distributed unevenly in the rat brain, and acute exposure to m‐xylene at a high concentration alters PSJTBPS binding, which may reflect changes in GABAA receptor characteristics.

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species.

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.

Allosteric Modulation of the GabaA Receptor In Rat Hypothalamus By Somatostatin Is Altered By Stress

1. This autoradiographic study was conducted to investigate somatostatin modulation of GABA(A) receptor binding in hypothalamic structures of immobilization-stressed rats. 2. GABA(A) receptor binding was labelled with [35S]-t- butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel. 3. Several structures of the rat hypothalamus (i.e. the peri- and paraventricular nuclei) display an increase in [35S]-TBPS binding as well as an alteration of the modulatory effect of somatostatin on the GABA(A) receptor complex under stress. Furthermore, these results demonstrate for the first time that somatostatin is particularly effective in modifying [35S]-TBPS binding to the GABA(A) receptor in rat hypothalamus.

Functional modulation of gamma-aminobutyric acid A receptors by etifoxine and allopregnanolone in rodents

We looked for an interaction between etifoxine and the neurosteroid allopregnanolone at central gamma-aminobutyric acid (GABA A) receptors. Etifoxine (2 μM) did not affect the affinity of allopregnanolone (IC 50=108 nM) for its site in preparations of Sprague–Dawley rat cerebral cortex membranes, as determined by the inhibition of [ 35S] t-butylbicyclophosphorothionate binding, a specific ligand of the GABA A receptor chloride channel site. Etifoxine and allopregnanolone were anticonvulsants, blocking the clonic convulsions induced by bicuculline (an antagonist of the GABA A receptor) in CD1 mice. A combination of subactive doses of the two compounds showed additive anticonvulsant effects. These results suggest that etifoxine and allopregnanolone bind to distinct putative recognition sites at or near the chloride channel site. Functionally, their binding may have an additive effect by enhancing GABA A inhibitory transmission.

Coupling between agonist and chloride ionophore sites of the GABA A receptor: agonist/antagonist efficacy of 4-PIOL

Eight γ-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA A receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and β-alanine on [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) binding to GABA A receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant α1β2γ2 (widespread major subtype) and α6β2γ2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [ 35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

Fipronil-Related Heterocyclic Compounds: Structure–Activity Relationships for Interaction with γ-Aminobutyric Acid- and Voltage-Gated Ion Channels and Insecticidal Action

To investigate the role of the heterocyclic moieties of nitrogen-containing phenyl heterocyclic compounds (PHCs) in interaction with γ-aminobutyric acid (GABA)-gated chloride channels, diverse classes of PHCs were examined for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive GABA antagonist, to housefly head and rat brain membranes. PHCs that inhibited [3H]EBOB binding include pyrazoles; 1H-1,2,3-triazoles; a 1H-1,2,4-triazole; 1,3,4-oxadiazol-2(3H)-ones; a 1,3,4-oxadiazole-2(3H)-thione; 1,2,4-oxadiazoles; a 1,2,4-thiadiazole; thiazoles; a 4(3H)-pyrimidinone; and a 2,4(1H,3H)-pyrimidinedione. An analogue (1) of the pyrazole insecticide fipronil, bearing an SCF3 group in place of the S(O)CF3, was found to be the most potent inhibitor with IC50s of 7.55 and 177 nM in housefly head and rat brain membranes, respectively. 3-(2,6-Dichloro-4-trifluoromethyl phenyl)-5-t-butyl-1,3,4-oxadiazol-2(3H)-one exhibited the highest selectivity for housefly GABA receptors versus rat receptors (IC50 rat/IC50 fly >204). PHCs that exhibited a comparable selectivity include a pyrazole and a 1H-1,2,3-triazole. Interaction of 16 selected PHCs with rat brain GABA-gated channels was also examined using [3S]t-butylbicyclophosphorothionate (TBPS), a radioligand for the mammalian noncompetitive antagonist site. A plot of pIC50s of 12 PHCs revealed a close correlation (r = 0.93) between their potency in inhibiting [3H]EBOB and [35S]TBPS binding. Scatchard analyses of the inhibition of [35S]TBPS binding by 1 suggested a competitive-type inhibition. A plot of the potency of 14 PHCs in inhibiting [3H]EBOB binding to housefly head membranes against their piperonyl butoxide-synergized insecticidal effect on German cockroaches yielded a close correlation (r = 0.89), with the exception of five triazoles and a 2,4(1H,3H)-pyrimidinedione. Although several selected PHCs also inhibited the specific binding of [3H]batrachotoxinin A 20-α-benzoate, a tritiated analogue of the sodium channel activator batrachotoxinin, to synaptosomes and membranes prepared from mouse brains, housefly heads, and American cockroach nerve cords, the concentrations required were higher than those of standard compounds producing significant effects on this system. The results demonstrate that a variety of five- and six- membered, nitrogen-containing heterocyclic compounds, bearing a 2,6-dichloro-4-trifluoromethyphenyl or 2,4,6- trichlorophenyl group, interact with ionotropic GABA receptors. Several PHCs display higher affinities for housefly GABA receptors and high selectivity as compared to rat GABA receptors. PHCs' insecticidal activity is mediated by their interaction with GABA-gated chloride channels.

Differential expression of GABA(A) receptor subunit mRNAs and ligand binding sites in rat brain following phencyclidine administration.

Recent biochemical observations have suggested the abnormalities in the gamma-amino-butyric acid (GABA)ergic system in schizophrenic brains. In the present study, we investigated the subunits gene expressions and ligand binding of the GABA(A) receptor following acute and chronic administration of phencyclidine (PCP), which induces schizophrenia-like symptoms, in rats using in situ hybridization and in vitro quantitative autoradiography. PCP i.p. administration at a daily dose of 7.5 mg/kg resulted in a significant decrease in expression of alpha 1 subunit mRNA in cerebral cortices (cingulate (-13%) and temporal cortex (-6%)) and hippocampal formation (CA1 (-11%), CA2 (-10%), CA3 (-11%) and dentate gyrus (-12%)) 1 h after a single treatment. In the repeated PCP administrations for 14 days, the expression of beta 2 mRNA in the cerebellum (-10%) and of beta 3 mRNA in the cerebral cortices (cingulate (-12%), parietal (-16%) and temporal cortex (-16%), caudate putamen (-18%), inferior colliculus (-18%), and cerebellum (-15%) were significantly decreased. In addition, [(35)S]t-butylbicyclophosphorothionate (TBPS) binding was also reduced in layer IV of the frontoparietal cortex (-14%), inferior colliculus (-17%), and cerebellum (-12%) following chronic PCP treatment, while no changes were observed following acute PCP treatment. These results indicate that single and repeated administrations of PCP independently regulate the expression of GABA(A)/benzodiazepine (BZD) receptor subunits mRNA and its receptor binding in the brain.

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.

Effect of Psychological Stress on [ 35S]TBPS Binding in Rat Brain

This study was designed to determine whether psychological stress alters the function of the GABAergic synapse, examined as biochemical changes of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) binding, in unwashed membranes of rat cerebral cortex. Psychological stress increased the number of [ 35S]TBPS binding sites by 22%. This enhancement was very similar to that after acute foot shock (24%). Psychological stress was induced very rapidly, because only 1 day after previous foot shock exposure, [ 35S]TBPS binding was increased by 23%. Diazepam [3 mg/kg intraperitoneally (subcutaneously)] and ipsapirone (5 mg/kg subcutaneously), injected 30 min before psychological stress, antagonized the enhancement of [ 35S]TBPS binding. This result suggests that psychological stress is a good animal model for investigating the various biochemical changes related to stress, avoiding the physical components associated with most of the normally used stressors and mimicking only emotional state alterations.