Jun-Ichi Taguchi

Stress-induced enhancement of suppression of [3H]GABA release from striatal slices by presynaptic autoreceptor

Abstract: The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (γ‐aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of γ‐aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas γ‐aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicucul‐line and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexame‐thonium and d‐tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, γ‐alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and γ‐aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.

Purification of γ-aminobutyric acid (GABA) receptor from rat brain by affinity column chromatography using a new benzodiazepine, 1012-S, as an immobilized ligand

The purification of gamma-aminobutyric acid (GABA) and benzodiazepine receptors from the rat brain was employed by the affinity column using a new benzodiazepine, 1012-S, as immobilized ligand. The 1012-S has a aliphatic primary amino group and exhibited an extremely high potency for displacing [3H]flunitrazepam binding to solubilized benzodiazepine receptor preparation (IC50 = 6.0 X 10(-11) M). This benzodiazepine affinity gel retained almost all of the solubilized GABA receptors from synaptic membranes applied to the column, and 25.6% of the receptor was eluted bio-specifically following the application of 1 mM 1012-S. The highest purification fold thus obtained was 4576 (specific activity: 0.99 nmol/mg protein). Furthermore, the successive application of 1-2 M NaSCN also resulted the elution of a highly enriched GABA receptor (specific activity: 0.41 nmol/mg protein; purification fold: 1889). SDS-polyacrylamide gel electrophoretic profiles of the bio-specifically eluted fraction with 1012-S showed the existence of two major bands having the molecular weights of approximately 48,500 and 54,500, in which the former band was selectively photoaffinity-labeled with [3H]flunitrazepam. On the other hand, it was found that the non-specifically eluted fraction with NaSCN contained 4 additional minor bands having molecular weights of 41,000 to 51,000. These results indicate that GABA receptor of the rat brain is coupled, at least in part, with benzodiazepine receptor and is readily purified by the use of highly specific benzodiazepine affinity gel, 1012-S-acetamide adipic hydrazide Sepharose 4B. The present results also suggest that the purified GABA/benzodiazepine receptor complex may contain two different kinds of subunits having the molecular weights of 48,000 and 54,500, in which the former subunit may possess benzodiazepine binding sites.

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.

Immunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABAA receptor complex

The distribution of the gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/Cl- channel complex in the rat brain was examined immunohistochemically using the specific antibody against purified GABAA receptor complex. The immunization of white albino rabbit with purified GABAA receptor complex resulted in the formation of specific antibody as indicated by the immunoprecipitation test. Immunohistochemical examinations using the antiserum on rat brain slices by the peroxidase-antiperoxidase method revealed the presence of the following immunoreactive sites which coincided with a previous report using antibody against L-glutamic acid decarboxylase; ventromedial nucleus of hypothalamus, red nucleus, globus pallidus, zona compacta and zona reticulata of substantia nigra, layers of Purkinje cells and granular cells of cerebellum, layers III-V of cerebral cortex and stratum radiatum of hippocampus. These results strongly suggest that immunohistochemical application of the antibody against the purified GABAA receptor complex is a useful tool for identifying GABAergic neurons having GABAA receptor complex-mediated synapses.

Functional coupling of γ-aminobutyric acid (GABA)a and benzodiazepine type II receptors: Analysis using purified GABA/benzodiazepine receptor complex from bovine cerebral cortex

Possible functional coupling between gamma-aminobutyric acid (GABA) and benzodiazepine receptors was examined using a purified GABA/benzodiazepine receptor complex. The purified receptor complex was obtained by 1012-S-acetamide adipic hydrazide Sepharose 4B affinity column chromatography, following the solubilization of synaptic membrane from the bovine cerebral cortex with Nonidet P-40. The binding of [3H]GABA to the purified GABA receptor was displaced significantly by muscimol and bicuculline, GABAA receptor agonists and antagonists, respectively, but not by baclofen, a GABAB receptor agonist. On the other hand, the binding of [3H]flunitrazepam to the purified benzodiazepine receptor was found to be displaced by microM ranges of CL 218,872, which is known to be sensitive to the benzodiazepine type II receptor. Furthermore, it was found that the binding of [3H]muscimol to these purified GABAA receptors was enhanced by benzodiazepines, while the binding of [3H]flunitrazepam to these benzodiazepine type II receptors was increased by GABA receptor agonists. These enhancements by GABA agonists and benzodiazepine agonists were found to be blocked by bicuculline and a benzodiazepine receptor antagonist, Ro15-1788, respectively. These results strongly suggest that the purified receptor may consist of GABAA and benzodiazepine type II receptors and possess a functional coupling of these sites, as shown in cerebral synaptic membranes.

Neuropeptide Participation in Canine Laryngeal Sensory Innervation: Immunohistochemistry and Retrograde Labeling

We investigated the quantitative participation of calcitonin gene-related peptide (CGRP), substance P (SP), and leu-enkephalin (ENK) in canine laryngeal sensory innervation by immunohistochemistry in combination with retrograde labeling using the recently introduced retrograde tracer cholera toxin subunit B—conjugated gold (CTBG). In the nodose ganglion, neurons labeled from the internal branch of the superior laryngeal nerve with CTBG were investigated immunohistochemically by means of antisera against CGRP, SP, and ENK. The percentages of neurons immunoreactive to each neuropeptide were as follows: CGRP 81.5%, SP 24.5%, and ENK 7.0%. These results suggest that CGRP is the main sensory neurotransmitter in canine laryngeal sensory innervation.

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.

Glycoprotein as a Constituent of Purified γ‐Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

Abstract: The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified γ‐aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydro‐chloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N‐ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cys‐tine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but SH and S‐S‐groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A‐Sepharose eluted protein having [H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glyco‐peptidase A, β‐galactosidase, and α‐mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with β‐galactosidase resulted in the disappearance of the low‐affinity site for [3H]muscimol binding and in an increase of Bmax of the high‐affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low‐affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with β‐galactosidase and glycopeptidase A were much higher than that with α‐mannosidase may also indicate a special importance of the β‐galactosyl residue in the inhibition of GABA receptor binding activity. Furthermore, the observation that the activation of high‐affinity [3H]muscimol binding by benzodiazepines disappeared following β‐galactosidase treatment suggests that the carbohydrate chain in the receptor complex may also be involved in the functional coupling between the GABA receptor and the benzodiazepine receptor.

Alcohol, acetaldehyde and salsolinol-induced alterations in functions of cerebral GABA/benzodiazepine receptor complex.

Effects of alcohol (ethanol) and acetaldehyde on the metabolism and function of primary cultured GABAergic neurons and that of salsolinol, a condensation product of acetaldehyde with dopamine, on cerebral GABA/benzodiazepine (BZP) receptor complex were investigated. In vitro addition of acetaldehyde induced a significant reduction not only on the content of GABA but also on the basal and GABA-activated [3H]flunitrazepam ([3H]FLN) bindings in primary cultured neurons. In contrast, alcohol exhibited only suppressive effects on [3H]FLN binding as well as on the enhancement of [3H]FLN binding by GABA. On the other hand, salsolinol showed a significant stimulatory effect on [3H]FLN binding to cerebral particulate fractions obtained from alcohol-untreated mice in the presence of NaCl. Although a similar activation of cerebral [3H]FLN binding by salsolinol was found in alcohol-treated mice, this activation was significantly weaker in alcohol-withdrawn mice than those found in alcohol-untreated as well as alcohol-inhaled mice. These results strongly suggest that acetaldehyde may have more important pathophysiological roles than those of alcohol in the exhibition of neurotoxicity during alcohol intake. The present results also suggest that salsolinol may have a modulatory role on cerebral benzodiazepine receptor and the decreased capacity of such a modulating mechanism may be involved in the exhibition of alcohol withdrawal syndrome, possibly by decreasing the function of endogenous ligands for benzodiazepine receptor in the brain.

Purification of γ-aminobutyric acid receptor, benzodiazepine receptor and C1 channel from bovine cerebral cortex by benzodiazepine affinity gel column chromatography

Purification of solubilized ?-aminobutyric acid (GABA) and benzodiazepine receptors by Nonidet P-40 from the bovine cerebral cortex was employed by the affinity column chromatography using a benzodiazepine, 1012-S, as immobilized ligand. Although this benzodiazepine affinity gel retained all of the solubilized benzodiazepine receptors from synaptic membranes applied to the column, 25.6% of the GABA receptor binding activity was recovered in the run-through fraction. After the washing with NaCl, the biospecific elution with 1 mM 1012-S resulted the elution of 31.3% of GABA receptor binding activity applied to the column. The highest purification fold thus obtained was 3789 (specific activity: 0.39 nmol/mg protein). Successive application of 1-2 mM NaSCN also resulted further elution of GABA receptor. Scatchard analysis of [(3)H]muscimol binding to these fractions indicated that the purified GABA receptor had high and low affinity binding sites as detected in solubilized and synaptic membrane fractions. In addition, it was found that the B(max) values for both binding sites in the 1012-S-eluted fraction increased significantly without changing the K(D) values. SDS-polyacrylamide gel electrophoretic profiles of the 1012-S-eluted fraction showed the existence of two major bands having the molecular weights of approx. 53,000 and 57,000, both of which were irreversibly photoaffinity-labeled with [(3)H]flunitrazepam. This result made a sharp contrast with the result obtained in the rat brain, in which the subunits of the purified receptor consisted of molecular weights of 48,500 and 54,500 and the former band was selectively photoaffinity-labeled with [(3)H]flunitrazepam. In contrast, the gel filtration using Sephadex G-200 of the purified GABA/benzodiazepine receptor complex from both animal species revealed the molecular weight of these receptor complexes was approx. 340,000. These results strongly suggest that the subunit structures of GABA/benzodiazepine receptors from the bovine and rat brains may be essentially different. On the other hand, the purification of GABA/benzodiazepine receptor complex and C1 channel solubilized by CHAPS, a non denaturating zwitterionic detergent, using the same benzodiazepine affinity column followed by DEAE-Sephacel ion exchange column chromatography indicated that the purified GABA receptor was functionally coupled with both benzodiazepine receptor and C1 channel. These results also indicate that GABA(A) receptor which is structurally coupled with benzodiazepine receptor and C1 channel is readily purified by the use of this benzodiazepine affinity gel, whereas GABA(B) receptor which is not associated with both components is recovered in its run-through fraction, respectively.