Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Reciprocal Innervation between Serotonergic and GABAergic Neurons in Raphe Nuclei of the Rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [³H] serotonin or [³H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA_A receptor agonist muscimol (3 to 30 M) and the GABA_B receptor agonist baclofen (30 and 100 M) inhibited [³H]serotonin and [³H]GABA release. These effects of muscimol were reversed by the GABA_A antagonists bicuculline (100 M). The GABA_B antagonist phaclofen (100 M) also antagonized the baclofen-induced inhibition of [³H]serotonin and [³H]GABA release. Phaclofen by itself increased [³H]serotonin release but it did not alter [³H]GABA overflow. Muscimol (10 M) and baclofen (100 M) also inhibited [³H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA_A and GABA_B receptors located on serotonergic neurons. The 5-HT_1A receptor agonist 8-OH-DPAT (0.01 to 1 M) and the 5-HT_1B receptor agonist CGS-12066A (0.01 to 1 M) inhibited the electrically stimulated [³H]serotonin and [³H]GABA release. The 5-HT_1A antagonist WAY-100135 (1 M) was without effect on [³H]serotonin and [³H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 M) did not alter the inhibitory effect of CGS-12066A (1 M) on [³H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 M) to reduce [³H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 M) still inhibited [³H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 M) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT_1A/B and GABA_A/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.     

Effect of flumazenil on GABAA receptors in isolated rat hippocampal neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

Clozapine's Antipsychotic Effects do not Depend on Blockade of 5-HT3 Receptors

Sixteen known 5-HT₃ receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, indicating that they are also GABA_A antagonists, some of them selective for subsets of GABA_A receptors. The 5-HT₃ receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT₃ receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [³⁵S]TBPS binding. Additivity experiments suggest that ten 5-HT₃ receptor blockers tested at low concentrations preferentially block subtypes of GABA_A receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA_A antagonist decribed here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severly decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(–)-zacopride as a GABA_A antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT₃ receptor blockers are due to selective antagonism of certain GABA_A receptors, and not to blockade of 5-HT₃ receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of 122 GABA_A receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA_A antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.     

Methylmercury modulates GABAA receptor complex differentially in rat cortical and cerebellar membranes in vitro

Effects of methylmercury (MetHg) on the specific [³H]flunitrazepam binding were studied in rat cortical and cerebellar P₂-fractions in vitro. MetHg did not affect significantly the specific [³H]flunitrazepam binding in unwashed P₂-fraction but increased it marginally (by 16%) at 100 M in washed P₂-fraction, in both brain regions.Muscimol (3 M), a GABA_A agonist, stimulated the [³H]flunitrazepam binding by 30% to 50% depending on the brain region. In washed cerebellar membranes the enhancing response of muscimol was 10 to 14% lower after preincubation of the tissue with MetHg but in cerebral cortex MetHg did not modulate the muscimol response at all. The results indicate that Met-Hg may have region specific effects on GABA_A receptors in vitro and the effect may depend on the occupational state of the GABA binding domain of the receptor complex.     

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.     

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. [ ³ H]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 M 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 M and 1.6 M using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 M, and 22 and 34%. 3. [ ³ H]MUS binding. H and M increased [³H]MUS binding to whole forebrain membranes about 3-fold with EC₅₀ values of 6.0 and 15 M. Using cerebellar membranes, H and M increased [³H]MUS binding ~68% with EC₅₀ values of 2.3 and 12 M, respectively. Scatchard analysis revealed that the enhancements of [³H]MUS binding were due primarily to increases in the number of binding sites (B_max values) with no effect on the high affinity binding constants (K_d values). The enhancing effect of H and M were not additive. 4. [ ³⁵ S]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 M, respectively. Using cerebellar membranes, the corresponding IC₅₀ values were 5.3 and 4.8 M. 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.     

Does gabapentin act as an agonist at native GABA<Subscript>B</Subscript> receptors?

Gabapentin, a novel anticonvulsant and analgesic, is a -aminobutyric acid (GABA) analogue but was shown initially to have little affinity at GABA_A or GABA_B receptors. It was recently reported to be a selective agonist at GABA_B receptors containing GABA_B1a-GABA_B2 heterodimers, although several subsequent studies disproved that conclusion. In the present study, we examined whether gabapentin is an agonist at native GABA_B receptors using a rat model of postoperative pain in vivo and periaqueductal gray (PAG) slices in vitro; PAG contains GABA_B receptors, and their activation results in antinociception. An intrathecal injection of gabapentin or baclofen, a GABA_B receptor agonist, induced antiallodynia in this postoperative pain model. Intrathecal injection of GABA_B receptor antagonists CGP 35348 and CGP 55845 antagonized baclofen- but not gabapentin-induced antiallodynia. In ventrolateral PAG neurons, baclofen activated G-protein-coupled inwardly rectifying K⁺ (GIRK) channels in a manner blocked by CGP 35348 or CGP 55845. However, gabapentin displayed no effect on the membrane current. In neurons unaffected by gabapentin, baclofen activated GIRK channels through GABA_B receptors. It is concluded that gabapentin is not an agonist at GABA_B receptors that are functional in baclofeninduced antiallodynia in the postoperative pain model in vivo and in GIRK channel activation in ventrolateral PAG neurons in vitro.     

Pretreatment of Rat Brain Synaptosomes with GABA Increases Subsequent GABA Uptake Via GABAB Receptor Activation

Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA_B receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA_B receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in V_max. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.     

Immunohistochemical localization of GABAA receptors in the scotopic pathway of the cat retina

The distribution of GABA_A receptors in the inner plexiform layer of cat retina was studied using monoclonal antibodies against the 2/3 subunits. A dense band of receptor labeling was found in the inner region of the inner plexiform layer where the rod bipolar axons terminate. Three forms of evidence indicate that the GABA_A receptor labeling is on the indoleamine-accumulating, GABAergic amacrine cell that is synaptically interconnected with the rod bipolar cell terminal. (1) Electron microscopy showed that the anti-GABA_A receptor antibody (62-3G1) labeled profiles that were postsynaptic to rod bipolar axons and made reciprocal synapses. (2) Indoleamine uptake (and the subsequent autofluorescence) combined with GABA_A receptor immunohistochemistry showed co-localization of the two markers in half of the receptor-positive amacrine cells. (3) Double labeling demonstrated that half of the receptor-positive somata also contained GABA. These results indicate that a GABAergic amacrine cell interconnected with the rod bipolar cell, most likely the so-called A17 amacrine cell, itself bears GABA_A receptors.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

The influence of ethanol on the functional status of GABA(A) receptors

Gamma aminobutyric acid (GABA) is one of the main inhibitory neurotransmitters in the mammalian brain. Its effects are realized via GABA_A, GABA_B, and GABA_C receptors. GABA_A is the most abundant type of GABA receptors. It consists of six classes of subunits, , , , , , and . Acute and chronic exposures to ethanol are accompanied by changes in structure and function of GABA_A receptors. These changes may be a basis for altered behavior seen in alcoholism.     

GABA A -receptors: Structural requirements and sites of gene expression in mammalian brain

GABA_A-receptors, the major synaptic targets for the neutotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABA_A-receptors are still elusive. Expression of cDNAs coding for the ₁- and ₁-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABA_A-receptors with fully functional benzodiazepine receptor sites were formed when the ₁- and ₁-subunits were coexpressed with the ₂-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (₁, ₂, ₃, ₅, ₆, ₁, ₂, ₃, ₂) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the ₁-, ₁- and ₂-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABA_A-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.Special issue dedicated to Dr. Erminio Costa     

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

The bovine striatal dopamine D₁ receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [³H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a K_d of 0.95 and 1.1 nM and a B_max of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [³H]SCH-23390 binding exhibited a clear D₁ dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent thancis-flupentixol in displacing [³H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[³H]SCH-23390 competition studies revealed the presence of a highaffinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40–80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obrained from the agonist-treated membrane. In both the preparations, 100 M guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.     

Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

GABA_A-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. At the light microscope level specific staining of GABA_A-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling of GABA_A-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations to be performed. Quantification of the labeling intensity revealed a higher concentration of GABA_A-receptors per m plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABA_A-receptors was observed. No specific labeling of GABA_A-receptors was observed in dissociated primary cultures of cerebellar astrocytes.Special issue dedicated to Dr. Eugene Roberts.     

GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam

Summary GABA_A receptors present on rat cerebellar granule cells in culture were studied by the whole cell patch clamp technique. Muscimol appeared to be more potent than GABA itself in activating Cl^– currents. A benzodiazepine, flunitrazepam, only slightly (10%) potentiated the GABA action.These results support the previous suggestion that GABA_A receptors containing the subunit, such as those in the cerebellum granule cells, are potently activated by muscimol. The present results also bear out the concept that GABA action on receptors containing the subunit is not potentiated by benzodiazepines.     

Changes of [3H]Muscimol Binding and GABAA Receptor β2-Subunit mRNA Level by Tolerance to and Withdrawal from Pentobarbital in Rats

Effects of continuous pentobarbital administration on binding characteristics of [³H]muscimol were examined by autoradiography, and levels of GABA_A receptor 2-subunit mRNA were investigated by in situ hybridization histochemistry in the rat brain. In order to eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. An experimental model of barbiturate tolerance and withdrawal was developed using i.c.v. infusion of pentobarbital (300 g/10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]muscimol binding were elevated in cingulate of frontal cortex (46%) and granule layer of cerebellum (32%) of rats 24-hr after withdrawal from pentobarbital, while it was only elevated in cingulate (58%) of tolerant rats. The GABA_A receptor 2-subunit mRNA was increased in the withdrawal rats only: in the cortex (9–14%), hippocampus (15–21%), inferior colliculus (21%), and granule layer of cerebellum (24%). These results show the involvement of GABA_A receptor and its 2-subunit up-regulations in pentobarbital withdrawal rats, and suggest that the levels of [³H]muscimol binding and GABA_A receptor 2-subunit mRNA are altered in a region-specific manner during pentobarbital withdrawal.     

GABAA receptor plasticity in Jurkat T cells

GABA_A receptors (GABA_AR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABA_AR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABA_AR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABA_AR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function.     

Do Antiepileptics Phenytoin,Carbamazepine, and Loreclezole Show GABAA Receptor Subtype Selectivity in Rat Brain Sections?

[³⁵S]t-Butylbicyclophosphorothionate ([³⁵S]TBPS), a convulsant site ligand of GABA_A receptors, was used in autoradiography with rat brain sections to test suggested receptor subtype-selective actions of antiepileptics phenytoin, carbamazepine and loreclezole on native GABA_A receptors. At maximal 100 M concentration, both phenytoin and carbamazepine decreased [³⁵S]TBPS binding only by 20%, indicating that their low potency and efficacy prevents their use as 1 subunit-identifying compounds. Ten M loreclezole did not affect the binding, but a further increase in loreclezole concentration strongly decreased it. The action of loreclezole, assumed to reflect 2/3 subunit-containing receptors, varied from brain region to region, but the effects were unrelated to the regional expression profiles of subunit variants. We conclude that in autoradiographic [³⁵S]TBPS binding assay neither carbamazepine, phenytoin nor loreclezole are useful tools in characterizing brain regional heterogeneity of GABA_A receptors in rats and that only loreclezole exhibits high, pharmacologically relevant efficacy.