Crude Synaptic Membranes Research Articles

Reversal of GABA-mediated Inhibition of the Electrically and Potassium Chloride Evoked [3H]-GABA Release from Rat Substantia Nigra Slices by DL-3-Hydroxy-3-Phenyl Pentanamide

The phenyl alcohol amides, DL-2-hydroxy-2-phenyl butyramide (CAS 52839-87-9), DL-3-hydroxy-3-phenyl pentanamide (CAS 131802-69-2, DL-HEPP) and DL-4-hydroxy-4-phenyl hexanamide (CAS 67880-30-2) and their fluorine and chlorine analogs, at a concentration of 100 micromol/L, did not displace [3H]-gamma-aminobutyric acid ([3H]-GABA, CAS 108158-36-7) from GABAA receptors and only weakly displaced [3H]-GABA and [3H]-CGP62349 (CAS 186986-97-0), a GABAB receptor antagonist, from GABAB receptors in rat brain crude synaptic membranes. The electrically and potassium chloride (15 mmol/L) evoked [3H]-GABA release in the presence of DL-HEPP, GABA and GABAB receptor ligands from rat brain substantia nigra (SN) slices was studied. R-Baclofen (CAS 69308-37-8) (10 micromol/L), a GABAB receptor agonist, produced an inhibition of the electrically evoked [3H]-GABA release and this inhibition was blocked by CGP 55845A (CAS 149184-22-5) (10 micromol/L), a GABAB receptor antagonist, but was not affected by DL-HEPP (100 micromol/L). CGP 55845A (10 micromol/L) did not alter the electrically evoked [3H]-GABA release in the absence of baclofen. The addition of DL-HEPP (100 micromol/L) alone did not affect the electrically-evoked release of [3H]-GABA release control, but it was able to significantly reduce the inhibitory effect of GABA (CAS 56-12-2) (10 micromol/L) on [3H]-GABA release evoked both by electrical and potassium chloride stimulation, in the presence of tiagabine (CAS 115103-54-3) (10 micromol/L), a GABA uptake blocker. In three preliminary experiments, bicuculline (CAS 485-49-4) (10 micromol/L) and picrotoxinin (CAS 17617-45-7) (10 micromol/L), two GABAA antagonists, inhibited the electrically evoked release of [3H]-GABA from rat SN slices, and DL-HEPP (100 micromol/L) reversed this inhibition. The mechanism of action of DL-HEPP is not known but, it might act as a negative GABA modulator in rat brain slices.

Diminished prevalence but preserved synaptic distribution of N-methyl- d-aspartate receptor subunits in the methyl CpG binding protein 2(MeCP2)-null mouse brain

In this study, we examined the prevalence and distribution of NMDA receptor subunits within crude synaptic membranes derived from the brains of mice lacking methyl CpG binding protein 2 (MeCP2). Our results show that the distribution of NMDA receptor subunits within the detergent soluble, detergent resistant, and postsynaptic density microdomains is preserved at MeCP2-null synapses. However, analysis of the NMDA receptor subunit expression revealed a decrease in the prevalence of the GluN1 and GluN2A subunits in MeCP2-null tissue. Collectively, these results indicate that synaptic membrane microdomains at synapses of the MeCP2-null brain develop normally, and that NMDA receptor subunits are properly targeted and distributed within them. The under-representation of the GluN1 and GluN2A subunits suggests that MeCP2-null synapses contain fewer mature NMDA receptor complexes, and raises the possibility that impaired NMDA receptor ontogeny could contribute to Rett syndrome pathophysiology.

Ca2+ Inhibits the Association of Memantine with N-Methyl-D-aspartate (NMDA) Receptor-Gated Ion Channels

We examined the effect of 1-amino-3,5-dimethyladamantane (memantine) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) on the inhibition of [(3)H]MK-801 binding to crude synaptic membranes of rat forebrains in the absence or presence of Ca(2+). Ca(2+) decreased the potency of memantine to inhibit [(3)H]MK-801 binding. The effect of Ca(2+) was apparently competitive with memantine and was not annulled by the addition of Mg(2+). Ca(2+) slightly enhanced [(3)H]MK-801 binding, but showed no effect on the displacement of [(3)H]MK-801 binding by MK-801. The Ca(2+)-sensitive interaction of memantine with N-methyl-D-aspartate (NMDA) receptor-gated ion channels may provide a clue to understanding its voltage-dependent and clinically tolerated character.

Increased 5-HT 2C receptor binding in the brain stem and cerebral cortex during liver regeneration and hepatic neoplasia in rats

In the present study, serotonin 2C (5-HT 2C) receptor binding parameters in the brainstem and cerebral cortex were investigated during liver generation after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatic neoplasia in male Wistar rats. The serotonin content increased significantly ( p < 0.01) in the cerebral cortex after PH and in NDEA induced hepatic neoplasia. Brain stem serotonin content increased significantly ( p < 0.05) after PH and ( p < 0.001) in NDEA induced hepatic neoplasia. The number and affinity of the 5-HT 2C receptors in the crude synaptic membrane preparations of the brain stem showed a significant ( p < 0.001) increase after PH and in NDEA induced hepatic neoplasia. The number and affinity of 5-HT 2C receptors increased significantly ( p < 0.001) in NDEA induced hepatic neoplasia in the crude synaptic membrane preparations of the cerebral cortex. There was a significant ( p < 0.01) increase in plasma norepinephrine in PH and ( p < 0.001) in NDEA induced hepatic neoplasia, indicating sympathetic stimulation. Thus, our results suggest that during active hepatocyte proliferation 5-HT 2C receptor in the brain stem and cerebral cortex are up-regulated which in turn induce hepatocyte proliferation mediated through sympathetic stimulation.

Effect of chronic ethanol exposure and its withdrawal on the endocannabinoid system

The present study investigated the effect of ethanol (EtOH) exposure and its withdrawal on the central endocannabinoid system utilizing an EtOH vapor inhalation model, which is known to produce functional tolerance and dependence to EtOH. Swiss Webster mice ( n = 24) were exposed to EtOH vapors for 72 h. Mice were sacrificed after 72 h following EtOH exposure ( n = 12) and 24 h after its withdrawal ( n = 12). Radioligand binding assays were performed to measure the density of CB 1 receptor and CB 1 receptor agonist-stimulated [ 35S]GTPγS binding in crude synaptic membranes isolated from the cortex, hippocampus, striatum and cerebellum. The density of CB 1 receptor was significantly decreased (31–39%) in all the brain regions when compared to the control group. The CB 1 receptor-stimulated G i/o protein activation was also found to be decreased (29–40%) in these brain regions of EtOH exposed mice. Recovery of the CB 1 receptor density, in addition to, the CB 1 receptor-mediated G-protein activation was observed after 24 h withdrawal from EtOH. The levels of cortical anandamide, which was significantly increased (147%) by EtOH exposure, returned to basal levels after 24 h of withdrawal from EtOH exposure. A significant reduction (21%) in the activity of fatty acid amide hydrolase was found in the cortex of EtOH administered mice. Taken together, the neuroadaptation in the EC system may have a potential role in development of tolerance and dependence to EtOH.

SAD: A Presynaptic Kinase Associated with Synaptic Vesicles and the Active Zone Cytomatrix that Regulates Neurotransmitter Release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Subcellular localization of neural‐specific NPDC‐1 protein

NPDC-1 is a gene specifically expressed in neural cells when they stop to divide and begin to differentiate. Immunocytochemical study analysis of differentiated PC12 cells transfected with NPDC-tag vectors showed that NPDC-1 is transported in vesicles from the Golgi apparatus to the cell membrane and is then likely internalized into endosomes. The protein colocalized, at least partially, with synaptic vesicle proteins: synaptophysin, synaptobrevin 2, and Rab3 GEP (Rab3 GTP/GDP exchange protein). Moreover, subcellular fractionation of rat brain showed that crude synaptic membrane and crude synaptic vesicle fractions were enriched in NPDC-1. Although NPDC-1 bound Rab3 GEP in vitro, it seems unlikely to be involved in Ca2+-dependent exocytosis and, thus, in synaptic vesicle trafficking.

3H]alpha,beta-methylene ATP binding to P2X purinoceptor is unaffected by volatile anaesthetics.

The P2X receptor is responsible for fast excitatory neurotransmission in the central nervous system. This receptor is suggested as one of the targets of volatile anaesthetics. The study was undertaken to examine the site of action of volatile anaesthetics, especially for P2X purinoceptor-mediated neurotransmission. The effect of sevoflurane and isoflurane on [3H]alpha,beta-methylene ATP binding was investigated in rat crude synaptic membranes. The crude synaptic membranes were prepared from Sprague-Dawley rat brains by centrifugation and then incubated with volatile anaesthetics or P2 receptor antagonists. [3H]alpha,beta-methylene ATP binding was unchanged by either sevoflurane or isoflurane at clinically relevant concentrations. Suramin, a P2 antagonist, significantly (P < 0.05) decreased the binding of [3H]alpha,beta-methylene ATP in a dose-dependent manner (68.7 +/- 14.7% at 10 micromol, 49.5 +/- 6.4% at 50 micromol, 24.3 +/- 5.7% at 100 micromol, n = 10, mean +/- SD), whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 antagonist, did not affect the binding. The suppressive effect of volatile anaesthetics on ATP-mediated excitatory synaptic transmission could be one site of action. However, the blockade of ATP binding to P2X receptors is not a mechanism of action of volatile anaesthetics.

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum.

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.

Effects of Ginseng Total Saponin on [3H]DAGO Bindings of Opioid μ-Receptors

Crude synaptic membrane fractions from the frontal cortex, striatum, brain stem and whole brain of rat were prepared to assay the effects of ginseng total saponin (GTS) on [³H]DAGO bindings of the opioid μ-receptors. Scatchard plots analysis binding data demonstrated that GTS (0.1 mg/ml) decreased the affinity of specific [³H]DAGO bindings without changes in Bmax in the frontal cortex and striatum. On the other hand, GTS did not affect the [³H]DAGO bindings in the brain stem and whole brain. These results suggest that the regulation of [³H]DAGO bindings by GTS may play roles in the change of the pharmacological responses of μ-opioids.

The modification of NMDA receptors by visual experience in the rat retina is age dependent

Extensive studies have shown that the activation of N-methyl- d-aspartate receptors (NMDARs) and the subsequent rise in the levels of postsynaptic calcium are critical events in the initiation of synaptic plasticity. Modification of the amount, or of the subunit composition of NMDARs, alters receptor function thereby affecting the development and/or efficacy of synaptic transmission. In the present study, a Western blot analysis was employed to investigate the effects of visual experience and age on the differential expression of NMDARs in the rat retina. A crude synaptic membrane fraction (SPM) was prepared and assayed with antibodies specific for either the NR1, NR2A or NR2B subunits. Relative to control animals raised in a diurnal light–dark cycle, a period of 1 week of dark-rearing caused an increase in the relative amount of NR1, a decrease in the level of NR2A, and no change in the level of NR2B subunit expression in postnatal day 12 rats. At 2 months of age, 1 week of dark-rearing had less effect, and at 6 months of age there was no difference between dark-reared and control animals. The effect of light exposure on dark-reared animals was tested for the 2-month-old animals. Light exposure for long periods (days), but not short periods (h), could reverse the dark-rearing effects. These data provide evidence for a developmentally regulated plasticity of NMDAR subunits in the retina.

Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse

BackgroundThe expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected.ResultsThe NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots. The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls. The GluR1 receptor subunit and its phosphorylation are simultaneously increased in retinae of the rdta mice.ConclusionsThese data indicate that the NMDA receptors and AMPA (GluR1) receptors are altered in the retinae of rdta mice that lack rod photoreceptors. Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin b 12 diet

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B 12 on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 μg vitamin B 12 per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B 12 ameliorates the second messenger-mediated injury.

Brain Insulin Receptors and Spatial Memory: CORRELATED CHANGES IN GENE EXPRESSION, TYROSINE PHOSPHORYLATION, AND SIGNALING MOLECULES IN THE HIPPOCAMPUS OF WATER MAZE TRAINED RATS

Evidence accumulated from clinical and basic research has indirectly implicated the insulin receptor (IR) in brain cognitive functions, including learning and memory (Wickelgren, I. (1998) Science 280, 517-519). The present study investigates correlative changes in IR expression, phosphorylation, and associated signaling molecules in the rat hippocampus following water maze training. Although the distribution of IR protein matched that of IR mRNA in most forebrain regions, a dissociation of the IR mRNA and protein expression patterns was found in the cerebellar cortex. After training, IR mRNA in the CA1 and dentate gyrus of the hippocampus was up-regulated, and there was increased accumulation of IR protein in the hippocampal crude synaptic membrane fraction. In the CA1 pyramidal neurons, changes in the distribution pattern of IR in particular cellular compartments, such as the nucleus and dendritic regions, was observed only in trained animals. Although IR showed a low level of in vivo tyrosine phosphorylation, an insulin-stimulated increase of in vitro Tyr phosphorylation of IR was detected in trained animals, suggesting that learning may induce IR functional changes, such as enhanced receptor sensitivity. Furthermore, a training-induced co-immunoprecipitation of IR with Shc-66 was detected, along with changes in in vivo Tyr phosphorylation of Shc and mitogen-activated protein kinase, as well as accumulation of Shc-66, Shc-52, and Grb-2 in hippocampal synaptic membrane fractions following training. These findings suggest that IR may participate in memory processing through activation of its receptor Tyr kinase activity, and they suggest possible engagement of Shc/Grb-2/Ras/mitogen-activated protein kinase cascades.

Low convulsive activity of a new carbapenem antibiotic, DK-35C, as compared with existing congeners

Since carbapenems and cephalosporins have been suggested to induce convulsive side effects through an inhibitory action on the central γ-aminobutyric acid (GABA)-mediated inhibitory transmission, the present study evaluated the convulsive activity of a new carbapenem antibiotic (1R,5S,6S)-6[(R)-1-hydroxyethyl]-2-[(3S,5S)-5(S-methyl-4-thiomorpholinylcarbonyl)pyrrolidin-3-thio]-1-methylcarbapen-2-em-3-carboxylic acid (DK-35C) in in vitro and in vivo experiments, in comparison with cefazolin, imipenem and meropenem. In in vitro experiments, their abilities to inhibit [ 3H]muscimol (5 nM) binding to GABA A receptors were measured using crude synaptic membranes prepared from the rat cerebral cortex. The concentrations (mM) of the antibiotics which inhibit 50% of the specific binding, were 0.6 for imipenem, 1.8 for cefazolin, 15.4 for DK-35C and 27.6 for meropenem. In in vivo experiments, intracerebroventricular (i.c.v.) injections of cefazolin, imipenem and DK-35C induced convulsions in a dose-dependent manner in rats. The doses (nmol/rat) of the antibiotics which induce convulsions in 50% of rats, were 57 for imipenem, 96 for cefazolin, 377 for DK-35C and >3000 for meropenem. In the mouse pentylenetetrazole (PTZ) convulsive model, intravenous pretreatment with cefazolin (800 mg/kg) or imipenem (200 mg/kg) shifted the dose–response curve of PTZ (i.p.) to the left, indicating enhancement of the convulsive activity of PTZ. However, pretreatment with cefazolin, meropenem or DK-35C at a dose of 400 mg/kg did not produce any marked effects on the convulsive activity of PTZ compared with the saline vehicle-pretreated control. The results clearly demonstrate a good correlation between in vitro GABA A receptor binding assay and in vivo i.c.v. convulsive model using rats, and suggest that DK-35C may possess a relatively weak convulsive activity mediated through an interaction with GABA A receptors.

Regulation of Calcium/Calmodulin-Dependent Protein Kinase II in the Adult Rat Retina is Mediated by Ionotropic Glutamate Receptors

This study is concerned with the transmitter-mediated regulation of the α(50 kDa) and β(60 kDa) subunits of calcium calmodulin dependent protein kinase II (CamKII) in the adult rat retina. The level of antibody binding to the CamKII and the activity of CamKII were found to be increased after intravitreal injection of glutamate. Changes in the levels of the antibody-binding to the subunits of CamKII were observed in different subcellular fractions of the retina with a maximum response observed in crude synaptic membrane fractions. The glutamate mediated increases in CamKII were specific and blocked by 3,5-Dimethyl-1 adamantanamine; 3,5-Dimethylamantadine (Memantine), (±) 2-Amino-5-Phosphopentonic (AP-5) and 6-Cyano-7-Nitroquinoxaline-2,3-Dione (CNQX) but not with dl -2-Amino-3-Phosphono-Propionic (AP-3). The results indicate that the retinal neurotransmitter, glutamate, can regulate retinal CamKII activity through ionotropic but not metabotropic glutamate receptors. NMDA-receptors were found to be necessary but insufficient to stimulate CamKII. A model in which cooperative interaction between NMDA and non-NMDA glutamate receptors/ion channels is presented to explain the glutamate stimulated increases in CamKII activity in the retina.

3H]-GABA binding to GABAA and GABAB sites on rat brain crude synaptic membranes.

3H]-GABA binding to GABAA and GABAB sites on rat brain crude synaptic membranes.

Clathrin-coated vesicles from bovine brain contain uncoupled GABA A receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [ 3H]muscimol, a GABA A-specific agonist, [ 3H]flunitrazepam, a benzodiazepine agonist, and [ 35S] t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA A receptor channels. Under standard conditions, the binding level of [ 3H]muscimol, [ 3H]flunitrazepam, and [ 35S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [ 3H]flunitrazepam binding site with a K D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [ 3H]flunitrazepam and [ 35S]TBPS binding assays. For [ 3H]flunitrazepam binding to synaptic membranes, GABA gave an EC 50=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [ 3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [ 35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA A receptor β2 and α1 subunits. Thus, in comparison to GABA A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA A receptor uncoupling.

An anaesthetic dose of ethanol inhibits cyclic AMP formation by altering the function of GS protein.

The effects of ethanol on cerebral gamma-aminobutyric acid (GABA)B receptor and its related signal transduction systems were studied. [3H]GABA binding to cerebral GABAB receptors in crude synaptic membranes from rat brain was unaffected by ethanol at concentrations up to 100 mM, although 100 mM ethanol suppressed not only the basal but also forskolin-stimulated cAMP formation in cerebral cortical membrane vesicles. The suppressive effect of baclofen and the stimulatory effect of forskolin on cAMP formation was also found to be unaltered by ethanol. Pretreatment of the membrane vesicles with islet-activating protein (IAP), which eliminates the function of Gi protein by its ribosylation, had no effect on the suppressive effects of ethanol on basal and forskolin-stimulated cAMP formation. In contrast, pretreatment of membrane vesicles with 'low pH', which is known to suppress the function of Gs protein, eliminated the suppressive effect of ethanol on cAMP formation. These results suggest that an anaesthetic dose of ethanol, which is below that necessary to cause changes in receptor binding, may not affect the functions of GABAB/Gi, G(O) protein/adenylate cyclase system, but suppresses cAMP formation systems which are coupled with Gs protein.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983.

Saturable binding of (±)‐[3H]‐baclofen and [3H]‐γ‐aminobutyric acid ([3H]‐GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. The binding of [3H]‐baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+‐dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. The saturable portion of bound [3H]‐baclofen formed 20.3 ± 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 μm), (–)‐baclofen (0.04 μm) and to a much lesser extent by (+)‐baclofen (33 μm). Isoguvacine, piperidine‐4‐sulphonic acid and bicuculline methobromide were inactive (up to 100 μm) and muscimol was only weakly active (IC50 = 12.3 μm). Saturable binding of [3H]‐GABA increased on adding CaCl2 or MgSO4 (up to 2.5 Mm and 5.0 Mm respectively) to the Tris‐HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline‐sensitive binding of GABA (GABAA site binding) and could be completely displaced by (–)‐baclofen (IC50 = 0.13 μm). Increasing the Ca2+ concentration (0 to 2.5 Mm) increased the binding capacity of the membranes without changing their affinity for the ligand. The binding of [3H]‐GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non‐ionic detergent Triton‐X‐100 (0.05% v/v) reduced the binding capacity by 50%. The pharmacological profile of displacers of [3H]‐GABA from GABAB sites correlated well with that for [3H]‐baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. It is concluded that [3H]‐baclofen or [3H]‐GABA are both ligands for the same bicuculline‐insensitive, divalent cation‐dependent binding sites in the rat brain.