Islet-Activating Protein Inhibits the β-Adrenergic Receptor Facilitation Elicited by γ-Aminobutyric AcidB Receptors

gamma-Aminobutyric acidB (GABAB) receptor recognition sites that inhibit cyclic AMP formation, open potassium channels, and close calcium channels are coupled to these effector systems by guanine nucleotide binding proteins (G proteins). These G proteins are ADP-ribosylated by islet-activating protein (IAP), also known as pertussis toxin. This process prevents receptor coupling to these G proteins. In slices of cerebral cortex and hippocampus from rat, stimulation of GABAB receptors with baclofen, a receptor agonist, also potentiates the accumulation of cyclic AMP stimulated by beta-adrenergic agonists. It was unknown whether those GABAB receptors that potentiate the beta-adrenergic response were also sensitive to IAP. IAP was injected intracerebroventricularly into rats to ADP-ribosylate IAP-sensitive G proteins. Four days after the IAP injection, 38% and 52% of these G proteins from cerebral cortex and hippocampus, respectively, were ADP-ribosylated by the IAP injection. In slices of both structures prepared from IAP-treated rats, the GABAB receptor-mediated potentiation of the beta-adrenergic receptor response was attenuated. Thus, many GABAB receptor-mediated responses are coupled to IAP-sensitive G proteins.     

Effect of Guanosine 5''-O-(3-Thiotriphosphate) and Calcium on γ-Aminobutyric AcidB Binding as a Function of Postnatal Development

We have examined the development of gamma-aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of approximately 50% with the GABAB ligand (-)-[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium- and guanosine triphosphate-binding protein (G protein)-regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTP gamma S was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptors affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTP gamma S may affect the GABAB receptor by different mechanisms.     

Effects of Dihydropyridine Calcium Channel Ligands on Rat Brain γ-Aminobutyric AcidB Receptors

This study shows that low nanomolar concentrations of the calcium channel antagonist nifedipine displaced [3H]baclofen labeling of gamma-aminobutyric acidB (GABAB) receptors, whereas similar concentrations of two calcium channel agonists stimulated this GABAB receptor labeling. Neither effect was likely to be due to dihydropyridine (DHP) binding to baclofen recognition sites, because the inhibitory ligand nifedipine primarily affected apparent receptor density rather than affinity. Although these results could reflect the coupling of GABAB receptors with calcium channels, they do not rule out other, possibly more direct interactions between GABAB receptors and DHP binding sites. These DHP effects occur at much lower concentrations and display other significant differences from previously reported effects of DHPs on other transmitter receptors.     

γ-Aminobutyric AcidB Receptor Activation Modifies Agonist Binding to β-Adrenergic Receptors in Rat Brain Cerebral Cortex

The interaction of isoproterenol with beta-adrenergic receptor (beta AR) binding sites was measured in membranes prepared from rat brain cerebral cortical slices previously incubated in the presence or absence of gamma-aminobutyric acid (GABA) receptor agonists. Both GABA and baclofen, but not isoguvacine, altered beta AR agonist binding by increasing the affinity of both the low- and high-affinity binding sites and by increasing the proportion of low-affinity receptors. The response to baclofen was stereoselective, and the effect of GABA was not inhibited by bicuculline. The results suggest that GABAB, but not GABAA, receptor activation modifies the coupling between beta AR and stimulatory guanine nucleotide-binding protein, which may in part explain the ability of baclofen to augment isoproterenol-stimulated cyclic AMP accumulation in brain slices.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Comparative Stereostructure-Activity Studies on GABAA and GABAB Receptor Sites and GABA Uptake Using Rat Brain Membrane Preparations

The affinities of a number of analogues of gamma-aminobutyric acid (GABA) for GABAA and GABAB receptor sites and GABA uptake were studied using rat brain membrane preparations. Studies on the (S)-(+)- and (R)-(-)-isomers of baclofen, 3-hydroxy-4-aminobutyric acid (3-OH-GABA), and 4,5-dihydromuscimol (DHM) revealed different stereoselectivities of these synaptic mechanisms in vitro. Although (S)-3-OH-GABA and, in particular, (S)-DHM were more potent than the corresponding (R)-isomers as inhibitors of GABAA binding, the opposite stereoselectivity was demonstrated for the GABAB binding sites. Thus, (R)-3-OH-GABA and (R)-baclofen were more potent than the (S)-isomers as inhibitors of GABAB binding, (R)-baclofen being some five times more potent than (R)-3-OH-GABA. These two (R)-isomers actually have opposite orientation of the substituents on the GABA backbones, suggesting that the lipophilic substituent of (R)-baclofen interacts with a structural element of the GABAB receptor site different from that that binds the very polar hydroxy group of (R)-3-OH-GABA. The O-methylated analogue of 3-OH-GABA, 3-methoxy-4-aminobutyric acid (3-OCH3-GABA), did not interact significantly with GABAB sites. The homologues of GABA, trans-4-aminocrotonic acid (trans-ACA), muscimol, and 3-OH-GABA, that is, 5-aminovaleric acid (DAVA), trans-5-aminopent-2-enoic acid, homomuscimol, and 3-hydroxy-5-aminovaleric acid (3-OH-DAVA), respectively, were generally much weaker than the parent compounds, whereas 2-hydroxy-5-aminovaleric acid (2-OH-DAVA) showed a significantly higher affinity for GABAB sites than the corresponding GABA analogue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the γ-Aminobutyric AcidA Receptor by γ-Aminobutyric Acid Levels Within the Postsynaptic Cell

Synaptosomes and synaptoneurosomes were prepared from rat cerebral cortex. Comparison of the amino acid levels in the two types of organelles and of the effects of gabaculine thereon indicated that the neurosome portion of synaptoneurosomes constituted the major influencing component of the organelles. Administration to rats of inhibitors of gamma-aminobutyric acid (GABA) degradation, such as gabaculine and L-cycloserine, resulted in elevated GABA levels in synaptoneurosomes and a decrease in muscimol-stimulated Cl- up-take by the organelles. Addition of gabaculine directly to the incubation medium for the uptake assay had no effect on the Cl- transport. In contrast, administration to rats of isonicotinic acid hydrazide, an inhibitor of GABA synthesis, decreased the GABA level in synaptoneurosomes and increased the muscimol-stimulated Cl- uptake by the organelles. Although the evidence is not unequivocal, it does support the concept of GABA released from nerve endings being taken up by the postsynaptic cell, from where it exerts a regulatory influence on the functioning of the GABA receptor/ion channel complex.     

Molecular Size of the γ-Aminobutyric AcidA Receptor Purified from Mammalian Cerebral Cortex

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.     

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Ethanol and the γ-Aminobutyric Acid-Benzodiazepine Receptor Complex

Abstract: Ethanol appears to enhance γ-aminobutyric acid (GABA)-mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA-benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 m M failed to alter binding of [³H]flunitrazepam (FNZ), [³H]Ro 15-1788, or [³H]methyl-γ-carboline-3-carboxylate (MBCC) to benzodiazepine receptors, or of [³H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 m M ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [³H]FNZ and/or [³H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA-benzodiazepine receptor complex.     

Bicuculline- and Baclofen-Insensitive γ-Aminobutyric Acid Binding to Rat Cerebellar Membranes

Up to 60% of gamma-[3H]aminobutyric acid ([3H]GABA) bound specifically to rat cerebellar membranes in the absence of Ca2+ was insensitive to the GABAA antagonist bicuculline and to the GABAB agonist baclofen. This indicates that a significant component of specifically bound [3H]GABA is associated with non-GABAA, non-GABAB binding sites. The presence of this binding component appeared seasonal, peaking in the month of September (early spring) each year over a 4-year period. The calcium independence and bicuculline and baclofen insensitivity of the binding indicate that this binding is not to the classical GABAA and GABAB binding sites. High concentrations of muscimol and isoguvacine inhibited non-GABAA, non-GABAB binding. Scatchard analysis of the non-GABAA, non-GABAB binding sites indicated two kinetic components: KD1 = 42 nM and KD2 = 9.2 microM; Bmax1 = 1.6 pmol/mg of protein and Bmax2 = 28 pmol/mg of protein.     

Ion and Temperature Effects on the Binding of γ-Aminobutyrate to Its Receptors and the High-Affinity Transport System

A set of procedures was developed to study the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na(+)-dependent transport carrier, at 25 and 37 degrees C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD less than 30 nM), middle- (KD = 100-500 nM), and low-affinity (KD greater than 5 microM) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.     

Avermectin B1a Modulation of γ-Aminobutyric Acid Receptors in Rat Brain Membranes

Avermectin B1a stimulates high-affinity binding of [3H]-gamma-aminobutyric acid (GABA) to receptors in washed rat brain membranes. Scatchard analysis of the data indicates that the drug does not significantly alter the apparent dissociation constant of GABA binding, but increases the detectable number of binding sites from 3.2 to 5.1 pmol/mg protein, (+)-Bicuculline completely blocks control and avermectin B1a-stimulated GABA binding, whereas picrotoxin antagonizes specifically the avermectin B1a-stimulated GABA binding. The avermectin B1a-stimulated GABA binding is also chloride ion-dependent, whereas GABA binding in the control is not. These observations suggest that the mechanism of avermectin B1a stimulation of GABA binding may involve the chloride ion channel.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Generation of Two Forms of the γ-Aminobutyric AcidA Receptor γ-2-Subunit in Mice by Alternative Splicing

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)