GABAA Receptors In Rat Brain Research Articles

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Monitoring in vivo and in real time the influence of selective antisense on GABAA receptors in rat brain

Monitoring in vivo and in real time the influence of selective antisense on GABAA receptors in rat brain

Differences Between Cerebrovascular and Anti-Ischemic Effects of Dopamine, Docosahexaenoyldopamine, and GABA–Docosahexaenoyldopamine Conjugate

The cerebrovascular and anti-ischemic effects of dopamine and synthetic docosahexaenoyldopamine (DHA-DA) and the GABA–DHA-DA conjugate (OXL1220) were compared and found to differ. Dopamine and DHA-DA increased blood flow in intact and ischemic brain that was associated with pronounced hypertensive reactions. The latter inhibited dopamine transporter with IC50 = 29 μM but was practically inactive as a ligand for dopamine receptors. OXL1220 produced a selective vasodilating effect on blood flow in brain subjected to transient global ischemia. Only OXL1220 competed in vitro for specific binding sites of [3H]-gabazine in rat-brain GABAA-receptors. Therefore, conjugation of a neuromediator (GABA) to the DHA-DA changed the dopaminergic activity of the compound to GABA-ergic with respect to vascular tone in ischemic brain.

Preclinical and clinical pharmacology of TPA023B, a GABAA receptor α2/α3 subtype-selective partial agonist

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABA(A) receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABA(A) subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABA(A) receptors as measured using an in vivo [(3)H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [(11)C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABA(A) receptor population for avoiding sedation in man.

Crystal structure of songorine chloride trihydrate, [C22H32NO3]Cl · 3H2O

C22H38ClNO6, trigonal, R3 (no. 146), a = 28.293(2) A, c = 7.4204(7) A, V = 5144.3 A, Z = 9, Rgt(F) = 0.051, wRref(F ) = 0.137, T = 296 K. Source of material Songorine, a C20-diterpenoid alkaloid is isolated from the chloroform extract of the roots ofAconitum szechenyianumGay, wetted by 10%ammoniumhydroxide solution before. The purified compound is dissolved in a 1% hydrochloric acid solution at first. Then the solution is adjusted to pH 6.5 with a 10% NaOH solution. Colorless single crystals were obtained at room temperature for days by slowly volatilizing the solvent. Experimental details Water H atoms were located in a difference Fourier map and refined with restrained O—H bond lengths (0.85(1) A) and fixed isotropic displacement parameters (0.08A). OtherH atomswere placed at calculated positions and refined using a riding model with O—H distances restrained to 0.82 A, C—H distances restrained to 0.93 A and N—H distance restrained to 0.91 A. Discussion Songorine belongs to the class of C20-diterpenoid alkaloids, a large group of biologically active compounds of naturally occurrence. As a diterpenoid alkaloid, songorine has been found to enhance both the orthodromic population spike and field excitatory postsynaptic potential (EPSP) in CA1 region of hippocampal slices [1]. Recently, it was indicated that songorine can enhance the excitatory synaptic transmission in rat hippocampus, and was proved that songorine is a novel non-competitive antagonist at the GABAA receptor in rat brain [2]. In the title crystal structure, there are one songorine cation, one chloride ion and 3 crystal water molecules (figure top). The bond length of C12—O2 is 1.202 A, and the angles of O2−C12−C11, O2−C12−C13 and C11−C12−C13 are 121.8(4)°, 122.8(4)° and 115.4(3)°, respectively, which indicates that the C12 and O2 are sp hybridized, and C11, C12, C13 and O2 atoms are in a plane. The bond length of C17—C16 is 1.322(6)A. The angles of C13− C16−C15, C17−C16−C15 and C17−C16−C13 are 108.0(3)°, 126.2(4)° and 125.5(4)°, respectively, which show an exomethylene attached to C16. The songorine cations are interlinked by hydrogen bonds (figure bottom). Intra-molecular hydrogen bonds O6W−H6WA···Cl1 and O5W−H5WA···Cl1 link the Cl−, crystal water O5W and O6W together. The hydrogen bonds C2−H2A···O4W, O1− H1···O4W, N1−H1N···O4W fix the crystal water O4W to songorine. The intermolecular hydrogen bonds O6W− H6WB···O5W (symmetric code: x,y,z−1) and C21−H21A···O1, C19−H19B···O1, C15−H15···O2 (symmetric code: x,y,z+1), link the songorinemolecules along [001]. The hydrogen bondsO5W− Z. Kristallogr. NCS 223 (2008) 111-113 / DOI 10.1524/ncrs.2008.0047 111 © by Oldenbourg Wissenschaftsverlag, Munchen

In vitro study of the interaction of Tilia europeae L. aqueous extract with GABAA receptors in rat brain

The interaction of GABAA receptor-complex in rat brain was investigated in vitro with aqueous extracts obtained from the inflorescences of Tilia europeae L., using the [3H]muscimol and [3H]flunitrazepam binding techniques to synaptic membranes and the uptake of 36Cl- to synaptoneurosomes from cortices. The extract inhibited [3H]muscimol binding, stimulated 36ClCl- uptake by synaptoneurosomes and displaced at high concentrations, the [3H]flunitrazepam bound to synaptic membranes. When analysed by HPLC, the aqueous extract of Tilia europeae L. contained several amino acids, including GABA (about 100 μM). This GABA content can justify the displacement of [3H]muscimol produced by the extract but it did not increase the binding of [3H]flunitrazepam, as expected. Probably the extract contains other benzodiazepine-like substances which displace the [3H]flunitrazepam binding and counteract the expected GABA-induced increase in [3H]flunitrazepam binding. © 1997 John Wiley & Sons, Ltd.

In vitro study of the interaction of Tilia europeae L. aqueous extract with GABAA receptors in rat brain

In vitro study of the interaction of Tilia europeae L. aqueous extract with GABAA receptors in rat brain

Inhibition of GABA-gated Chloride Channels in Brain by the Arachidonic Acid Metabolite, Thromboxane A2

Previously, we showed that arachidonic acid and prostaglandin metabolites inhibited GABAA responses in rat cerebral cortex. Thromboxane A2 (TXA2), a metabolite of arachidonic acid, has potent actions on blood vessels and platelets, but its actions on neurons are not known. Here, we examined the effects of several TXA2 analogs on the functional and binding characteristics of GABAA receptors in rat brain. The stable analogs of TXA2, pinane and carbocyclic TXA2, and the TXA2 agonist, U-46619, inhibited muscimol-induced 36Cl− uptake in cerebral cortical synaptoneurosomes. Carbocyclic TXA2 decreased the maximal response to muscimol, consistent with a non-competitive interaction. The TXA2 antagonist, SQ 25,548, did not block the effects of either arachidonic acid or carbocyclic TXA2. Neither the biologically inactive metabolite of TXA2, TXB2, nor carbacyclin, a stable analog of prostacyclin (prostaglandin I2) had an effect on GABAA responses. Thus the pharmacology differs from that in vascular smooth muscle and platelets. To determine if GABAA receptors were sensitive to the thromboxanes, the effect of pinane TXA2 on the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to GABA-gated Cl− channels was measured using receptor autoradiography. Pinane TXA2 inhibited [35S]TBPS binding in a regionally selective and non-competitive manner; the greatest inhibition was in the cerebral cortex, hippocampus and striatum, areas which are selectively vulnerable to cerebral ischemia. We conclude that TXA2 can interact with neuronal membranes to inhibit GABA receptor function, independent of its actions on the cerebrovasculature and on glial cells. This may be important during pathologic states such as ischemia, when TXA2 accumulates in extracellular spaces. Copyright © 1996 Elsevier Science Ltd.

Effects of chlorotrifluoroethylene oligomer fatty acids on recombinant GABA receptors expressed in Xenopus oocytes.

GABA-activated Cl- current was expressed in Xenopus oocytes after injecting cRNA that had been transcribed in vitro from complementary DNA (cDNA) coding for a single GABA rho i-subunit cloned from human retina. The expressed current was insensitive to 100 microM bicuculline, but was activated by the GABA analogue trans-4-aminocrontonic acid (TACA). Anion-selective permeability of the expressed rho 1-subunit was determined by isotonically replacing the extracellular Cl- with different anions. The anion permeability was very similar to the native GABAA receptor/channel following a sequence of SCN- > I- > NO3- > Br- > or = Cl-. Halogenated fatty acids, such as chlorotrifluoroethylene (CTFE) and perfluorinated oligomer acids inhibited the GABA-induced current in oocytes expressing the human retinal GABA rho 1-subunit or rat brain GABAA receptor alpha 1,beta 2,gamma 2 subunits. The inhibitory effect of halogenated fatty acids demonstrated a carbon chain length-dependent manner of: C10 > C8 > C6 > C4. Perfluorinated C8-oligomer acid (PFOA) was less effective at blocking this channel than the C8-CTFE oligomer acid. Radiolabeled GABA binding assay indicated that CTFE oligomer acids do not interfere at the GABA binding site of the receptor. Furthermore, the C8-CTFE oligomer fatty acid did not compete with picrotoxin for binding sites within the pore of the channel. These studies demonstrated that the heterologous expression system is useful for studying the molecular interaction between potential neurotoxic agents and neuroreceptors. Our results provide detailed information that should contribute to our understanding of the structure and function of retinal GABA receptors.

Distribution of [3H]zolpidem binding sites in relation to messenger RNA encoding the α1, β2 and γ2 subunits of GABAA receptors in rat brain

Localization of the messenger RNAs that encode the alpha 1, beta 2 and gamma 2 subunits of GABAA showed a distinct topographic pattern in rat brain which corresponded with [3H]zolpidem binding in most brain regions. The close topographic correspondence between the specific receptor subunits examined and the distribution of [3H]zolpidem binding sites provides support for the hypothesis that this benzodiazepine type 1 selective ligand binds to a GABAA receptor that consists of alpha 1, beta 2 and gamma 2 subunits in the rat brain. Brain regions with relatively high densities of alpha 1, beta 2 and gamma 2 subunits of GABAA and [3H]zolpidem binding included olfactory bulb, medial septum, ventral pallidum, diagonal band, inferior colliculus, substantia nigra pars reticulata and specific layers of the cortex. Two areas with low [3H]zolpidem binding and a virtual absence of these GABAA receptor subunit messenger RNAs were the lateral septum and the striatum. In contrast to the discrete pattern observed for alpha 1 and beta 2 subunit messenger RNAs, the gamma 2 subunit messenger RNA was distributed more diffusely in brain. Only the hippocampus, layer 2 of the piriform cortex and the cerebellum showed a strong concentration of the gamma 2 subunit messenger RNA. It was determined with a polymerase chain reaction assay that both long and short variants of the gamma 2 subunit messenger RNAs were present within several of the brain sites selected for examination. Sites with high densities of [3H]zolpidem binding sites had a greater relative abundance of the gamma 2 long splice variant, compared to the gamma 2 short variant. There were some regions that expressed high levels of alpha 1, beta 2 and gamma 2S subunit messenger RNAs but low [3H]zolpidem binding, suggesting that gamma 2 splice variant expression may modulate high-affinity [3H]zolpidem binding. To determine relationships between in vitro [3H]zolpidem binding and functional sensitivity in vivo, interactions between zolpidem and GABA were assessed in brain regions that contained high and low densities of [3H]zolpidem binding sites. In the medial septum, a brain region with a high concentration of [3H]zolpidem binding sites, iontophoretic application of zolpidem enhanced the inhibitory effect of GABA responses on 70% of the neurons examined. In the lateral septum, which contains very low densities of [3H]zolpidem binding sites, neurons were not sensitive to zolpidem enhancement of GABA-induced inhibition. These electrophysiological results demonstrate a correspondence between the regional distribution of [3H]zolpidem binding in vitro and functional sensitivity to the drug in vivo.

Sesquilignan Haedoxans: Interaction with the GABAAReceptor in Rat Brain

(+)-Haedoxan A, an insecticidal sesquilignan, inhibited the specific binding of the noncompetitive GABA antagonist [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes in concentration-dependent and noncompetitive manners. The data show haedoxan’s interaction with a site coupled to the TBPS-binding site on the GABAA receptor in rat brain, while the physiological significance of the interaction remains to be discovered.

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.

The γ3-subunit of the GABA A-receptor confers sensitivity to benzodiazepine receptor ligands

The γ3-subunit of the GABA A-receptor in rat brain has been identified by molecular cloning. When co-expressed with the α5- and β2-subunits in transfected cells a high potency for GABA ( K a = 4.9 ± 1.2 μM; and a strong cooperativity in gating the channel ( H = 1.9 ± 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam reduced by βCCM. An analogous bi-directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations α1β2γ3 and α3β2γ3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the γ3-subunit, as tested with the α3β2- and α5β2-subunit combinations, the γ3-subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABA A-receptors. The γ3-subunit could functionally replace the γ2-subunit with regard to the bi-directional allosteric drug modulation.

O-302 - Comparative study of agonist and antagonist binding to GABAA receptors in rat brain

O-302 - Comparative study of agonist and antagonist binding to GABAA receptors in rat brain

Functional expression and sites of gene transcription of a novel α subunit of the GABA A receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits ( K a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA A receptors in neuronal populations within the olfactory bulb.

Actions of the insecticide 2 (nitromethylene) tetrahydro-1, 3-thiazine on insect and vertebrate nicotinic acetylcholine receptors

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10(-4) M) and alpha-bungarotoxin (1.0 x 10(-7) M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAA receptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibit an agonist action at nicotinic acetylcholine receptors.

Action of Organophosphates on GABAA Receptor and Voltage-Dependent Chloride Channels

The effects of several organophosphates were studied on the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to rat brain GABAA receptor and receptor function as assayed by GABA-induced 36Cl− influx into membrane vesicles and on the binding of [35S]TBPS to a voltage-dependent Cl− channel in Torpedo californica electric organ. The organophosphate anticholinesterases diisopropylphosphorofluoridate, soman, sarin, tabun, and VX had little or no effect on GABA-regulated chloride channels. They also had no effect on [35S]TBPS binding to the voltage-dependent chloride channel, except for soman which inhibited it with an IC50 of 24 μM. Triphenyl phosphate was the only one of three organophosphate flame retardants tested that inhibited both GABA-regulated chloride channel and binding of [35S]TBPS to the voltage-dependent chloride channel with IC50s of 18 and 13 μM, respectively. The industrial organophosphate tri-o-cresyl phosphate and the anticholinesterase organophosphate insecticides leptophos, leptophos oxon, and O-ethyl O-4-nitrophenyl phenylphosphonothioate inhibited GABA-regulated chloride channels and bound with high affinity to the voltage-dependent chloride channels (lC50 = 0.3 to 8.7 μM). There was no apparent correlation between the affinities of the GABAA receptor chloride channel or the voltage-dependent chloride channel for the different organophosphates and their potencies in inhibiting acetylcholinesterase or in inducing delayed neurotoxicity. Nevertheless, although the voltage-dependent chloride channel and/or GABAA receptor are not primary targets for organophosphate anticholinesterases and flame retardants, it is suggested that the inhibition of these two proteins by certain organophosphates may contribute to their toxicities.

Effects of insecticides on GABA‐induced chloride influx into rat brain microsacs

The actions of different insecticides known to affect binding of ligands to gamma-aminobutyric acid (GABA) receptors were studied on the function of GABAA receptors in rat brain as assayed by GABA-induced 36Cl- influx into membrane microsacs. This flux was inhibited by the competitive antagonist bicuculline and the noncompetitive antagonist t-butylbicyclophosphorothionate, and the GABA effect was potentiated by the tranquilizer flunitrazepam and the depressant pentobarbital, as expected for effects on a GABAA receptor. The GABA-induced 36Cl- flux was inhibited by several cyclodienes and gamma-hexachlorocyclohexane (gamma-BHC) with the following order of decreasing potency: endosulfan I greater than endrin greater than endosulfan II greater than dieldrin greater than heptachlor epoxide greater than gamma-BHC greater than heptachlor. The noninsecticidal beta-BHC had no effect, while the IC 50 values for gamma-BHC and endrin were 1 and 0.2 microM, respectively. The four pyrethroids tested also inhibited the GABA-induced 36Cl- flux with the following decreasing potencies: 1R,cis,alpha S-cypermethrin greater than 1R,trans, alpha S-cypermethrin greater than fluvalinate greater than allethrin. Avermectin B1a was the only insecticide tested that, in the absence of GABA, stimulated 36Cl- flux in a dose-dependent manner, and this flux was inhibited by bicuculline. The stereospecific inhibition of the GABA-induced 36Cl- influx by the cyclodienes and gamma-BHC supports previously published data on their binding to mammalian brain GABAA receptor and suggests that these insecticides inhibit this receptor's function. It is also suggested that type II pyrethroids are potent inhibitors of the same receptor. However, avermectin B1a appears to act as a partial agonist of GABAA receptors.