Masaaki Hirouchi

Functional Coupling of the γ-Aminobutyric AcidB Receptor with Calcium Ion Channel and GTP-Binding Protein and Its Alteration Following Solubilization of the γ-Aminobutyric AcidB Receptor

Abstract: The coupling mechanism of the γ‐Aminobutyric acid (GABA)B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP‐binding protein was examined using a crude synaptic membrane (P2) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P2 fraction, [3H]GABA binding to the GABAB receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), a calmodulin antagonist, did not affect on the calcium ion‐induced enhancement of GABAB receptor binding. These results suggest that the GABAB receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABAB receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5′‐(3‐O‐thio)triphosphate (GTPγS), guanosine 5′‐(β,γ‐imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (‐)‐baclofen. Although the affinity of (‐)‐baclo‐fen for the GABAB receptor was decreased in the presence of GTP, pretreatment of the P2 fraction with islet‐activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (‐)‐baclofen induced an increase of GTPase activity in the P2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABAB receptor may also be functionally coupled with IAP‐sensitive GTP‐binding protein. Treatment of the P2 fraction with sodium deoxycholate resulted in the highest solubilization of GABAB receptor among various detergents examined. The solubilization, however, completely eliminated the stimulating effects of calcium ion and calcium ion channel blockers as well as the inhibitory effects of GTP and GTP analogues on GABAB receptor binding. Furthermore, the increase of GTPase activity induced by GABA and (‐)‐baclofen was also eliminated following the solubilization. These results suggest that functional coupling of the GABAB receptor with the calcium ion channel and GTP‐binding protein such as Ni or No may be easily destroyed following the solubilization.

Muscimol-induced reduction of GABAA receptor α1-subunit mRNA in primary cultured cerebral cortical neurons

The expression of mRNA for GABAA receptor alpha 1-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABAA receptor. The level of mRNA for GABAA receptor alpha 1-subunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of beta-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor alpha 1-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABAA receptor, and muscimol. The expression of mRNA for GABAA receptor alpha 1-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABAA receptor complex may induce the reduced expression of mRNA for the receptor complex.

Structure and function of cerebral GABAA and GABAB receptors

The receptor for GABA (gamma-aminobutyric acid), an inhibitory neurotransmitter in the brain, has been classified into GABAA and GABAB types. The GABAA receptor was purified by means of affinity column chromatography using benzodiazepine as an immobilized ligand. The results indicated that the GABAA receptor consists of several subunits and forms a GABA-gated Cl- channel, which is coupled with the benzodiazepine receptor. The molecular weight of the GABAA receptor complex was estimated to be approximately 300 kDa. Furthermore, cDNA cloning of GABAA receptor subunits was performed and the primary structure of these subunits was deduced. The results suggested that these subunits possess four transmembrane domains in their structure which are important for the formation of the Cl- channel. On the other hand, activation of GABAB receptors induced the inhibition of adenylyl cyclase activity and phosphatidylinositol turnover via inhibitory GTP-binding proteins such as G(i) and/or G(o). The GABAB receptor was purified using baclofen affinity and immunoaffinity column chromatographies. It was confirmed that the purified GABAB receptor protein is about 80 kDa in its molecular weight. This protein is capable of inducing the inhibition of adenylyl cyclase when it is reconstituted with G(i)/G(o) protein in the phospholipid vesicle system. Currently available data indicate that GABAA and GABAB receptors in the central nervous system are distinct not only in terms of their molecules but also their signal transduction systems. However, the primary structure and synaptic localization of GABAB receptor molecules in the brain remain to be clarified.

Functional coupling of Gi subtype with GABAB receptor/adenylyl cyclase system: analysis using a reconstituted system with purified GTP-binding protein from bovine cerebral cortex.

A single molecular species of GTP-binding protein (G protein) has been purified from the bovine cerebral cortex. The immunoblot analysis indicated that the isolated G protein might be Gi1 or Gi2 but not Go, since it was reacted by specific antibodies, anti-Gi alpha 1-2 and anti-Gi alpha 1-1, but not anti-Go alpha. When the Gi protein was reconstituted into phospholipid vesicles with partially purified GABAB receptor and adenylyl cyclase, the stimulation of GABAB receptor by its agonists induced the inhibition of forskolin-stimulated cAMP accumulation. This GABA-induced inhibition was abolished by CGP 55845A, an antagonist of GABAB receptor. These results suggest that a Gi subtype, which was suggested to correspond to Gi1 or Gi2 may be functionally coupled with GABAB receptor/adenylyl cyclase system.

Alteration of GABAA receptor α1-subunit mRNA in mouse brain following continuous ethanol inhalation

Alterations in the expression of mRNA for GABAA receptor alpha 1-subunit were analyzed in the brain using mice that had been made alcohol-dependent, and exhibited a decrease in GABA-dependent 36Cl- influx into membrane vesicles following continuous ethanol inhalation for 7 days. Continuous ethanol inhalation for more than 5 days induced a significant increase in the expression of GABAA receptor alpha 1-subunit mRNA in the brain without significantly altering total poly(A)+ RNA content. Furthermore, it was found that the increase in expression of GABAA receptor alpha 1-subunit mRNA in the brain turned to its normal level 8 h after the ethanol inhalation was terminated. In contrast, the expression of beta-actin mRNA in the brain was not altered under the same experimental conditions. These results suggest that continuous ethanol inhalation may induce not only the suppression of the functions of GABAA receptor complex but also a reactive increase in the expression of mRNA for GABAA receptor subunits in the brain.

GABA-stimulated 36Cl− influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.

Nucleotide and deduced amino acid sequences of the GABAA receptor α-subunit from human brain

cDNA cloning of the ?-aminobutyric acid (GABA)(A) receptor ?-subunit from human brain has been attempted in order to clarify its structure. Using a bovine cDNA fragment as a homologous probe, the cDNA clone containing complete coding regions of the GABA(A) receptor ?-subunit was isolated by screening the human brain cDNA library, and its full amino acid sequences were deduced from the nucleotide sequence. The nucleotide and deduced amino acid sequences showed a high homology in comparison with those in bovine brain. The human brain mRNA indicated the presence of a single mRNA species of about 4500 bases by RNA blot hybridization. In addition, Southern blot analysis suggested that the human gene of the GABA(A) receptor ?-subunit might possess an exon-intron organization.

Role of metabotropic glutamate receptor subclasses in modulation of adenylyl cyclase activity by a nootropic NS-105.

The involvement of metabotropic glutamate (mGlu) receptors in the modulatory actions of a novel cognition enhancer, (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105), on adenylyl cyclase activity in rat cerebrocortical membranes and primary neuronal cultures was investigated using selective antagonists and antisense oligodeoxynucleotides for mGlu receptor subclasses. In rat cerebrocortical membranes, the inhibitory action of NS-105 (0.1 microM) on forskolin-stimulated cAMP formation was blocked by a group II mGlu receptor antagonist, (+/-)-alpha-ethylglutamic acid, and by a group III antagonist, (+)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP-4), but not by a group I antagonist, (+/-)-1-aminoindan-1,5-dicarboxylic acid (AIDA), whereas the facilitation of cAMP formation by NS-105 (1 microM) in pertussis toxin-pretreated membranes was abolished by AIDA but not by (+/-)-alpha-ethylglutamic acid or MAP-4. In primary cultured neurons of mouse cerebral cortex, the inhibitory action of NS-105 on adenylyl cyclase activity disappeared after treatment with antisense oligodeoxynucleotides for group II (mGlu(2) and mGlu(3) receptors) and group III (mGlu(4) and mGlu(7) receptors) but not group I (mGlu(5) receptor) mGlu receptor subclasses. These findings suggest that the inhibitory action of NS-105 on adenylyl cyclase activity is mediated through group II and group III mGlu receptor subclasses while the facilitatory action is dependent on the group I mGlu receptor subclass.

Expression of β-adrenergic receptor up-regulation is mediated by two different processes

Mechanisms of up-regulation of beta-adrenergic receptors (beta-ARs) induced by sustained exposure to 10(-8) M nadolol, a non-selective beta-AR antagonist, were examined using mouse cerebrocortical neurons. Nadolol dose- and time-dependently increased [3H]CGP-12177 bindings to the particulate fractions. This increase occurred 6 h and attained its plateau 12 h after the exposure, whereas beta1- and beta2-AR mRNA significantly increased 24 h and attained their plateaus 3 days after the exposure. Scatchard analysis revealed that the increased bindings were due to increase of receptor density. The [3H]CGP-12177 bindings to beta1- and beta2-ARs increased both 12 h and 5 days after the exposure. Although cycloheximide (CHX) decreased the bindings with or without nadolol, the extent of increase of the bindings induced by nadolol was not affected by CHX. Actinomycin D (AD) with nadolol showed no affects on the bindings 12 h after nadolol exposure, while AD treated 6 h after nadolol exposure significantly reduced the bindings 48 h after nadolol exposure. During 24 h after nadolol exposure, the increase in proteins of beta1- and beta2-ARs in the neuronal membrane was due to the increased receptor protein translocation from cytosol to membrane. These results indicate that the up-regulation of beta-ARs induced by nadolol is mediated by, at least, two different processes, one is increase in translocation of receptor proteins from cytosol to membrane with no changes in synthesis of receptor proteins and their mRNA and another is dependent on receptor protein synthesis with increased synthesis of their mRNA.

Effects of thyroxine and its related compounds on cerebral gaba receptors: Inhibitory action of benzodiazepine recognition site in GABAA receptor complex

The effects of thyroxine and its related derivatives on gamma-aminobutyric acid (GABA) receptors in the rat brain were examined. D-Thyroxine strongly inhibited [3H]flunitrazepam binding to benzodiazepine receptor in crude synaptic membrane from the rat brain. The Scatchard analysis of the [3H]flunitrazepam binding in the presence of D-thyroxine indicated the decreases in the affinity and maximum number of binding site. Furthermore, D-thyroxine inhibited the enhancing effect of flunitrazepam on GABA-stimulated 36Cl- influx into membrane vesicles, although GABA-stimulated 36Cl- influx alone was not affected by D-thyroxine. On the other hand, the effects of thyroxine and its related derivatives on cerebral GABAB receptor binding were not noted. These results suggest that D-thyroxine may be a drug which is able to modulate the function of GABAA receptor complex via the inhibitory action on benzodiazepine recognition site.