The Level of GAD67 Protein Is Highly Sensitive to Small Increases in Intraneuronal γ-Aminobutyric Acid Levels

Increases (>2.5-fold) in GABA levels in rat brain lead to a large decrease in the level of the 67-kDa form of glutamate decarboxylase (GAD₆₇) through a mechanism involving either a change in GAD₆₇ protein stability or a change in GAD₆₇ mRNA translation. In the present study, brain levels of GABA were manipulated by treating rats with various doses of γ-vinyl-γ-aminobutyric acid (GVG), and the dependence of total GAD activity and levels of GAD₆₇ and GAD₆₅ protein on the levels of GABA was analyzed. Initial studies showed that both GABA and GAD₆₇protein levels reached new steady-state levels after two to four daily injections; GABA increased 1.5- (30 mg of GVG/kg) and fourfold (150 mg of GVG/kg), and GAD₆₇ protein content decreased by 30 and 70%. To assess the sensitivity of GAD₆₇ to GABA, rats were injected with eight different doses of GVG (15-150 mg/kg) for 5 days. With increasing doses of GVG, we observed a gradual increase in both whole-tissue and synaptosomal GABA levels and a gradual decrease in GAD₆₇ protein and GAD activity. The levels of GAD₆₇ remained constant at all GVG doses. GAD₆₇ was remarkably sensitive to GABA. The synaptosomal GAD₆₇ level decreased ∼12% and the whole-neuron GAD₆₇ level decreased ∼3% for each 1 % increase in nerve terminal GABA content when it was close to its physiological level. Our results clearly demonstrate that GAD₆₇ is tightly controlled by intraneuronal GABA, and we suggest that this regulatory mechanism has important implications for the physiological regulation of GABAergic function in the mammalian brain.     

Effects of Increased γ-Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex

Abstract: Rats were injected with saline or the γ-aminobutyric acid (GABA) transaminase inhibitor γ-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD₆₇ and GAD₆₅) in the cerebral cortex were studied. γ-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75–80% decrease in GAD₆₇ levels, whereas the levels of GAD₆₅ remained unchanged. RNA slot-blotting with a ³²P-labeled GAD₆₇ cDNA probe demonstrated that the change in GAD₆₇ protein content was not associated with a change in GAD₆₇ mRNA levels. Our results suggest that GABA specifically controls the level of GAD₆₇ protein. This effect may be mediated by a decreased translation of the GAD₆₇ mRNA and/or a change in the stability of the GAD₆₇ protein.     

Striatal Expression of Glutamic Acid Decarboxylase Gene in Alzheimer's Disease

Abstract: To examine potential alteration of GABAergic striatal neurons in Alzheimer's disease, we used quantitative in situ hybridization to analyze the messenger RNA coding for M_r 67,000 glutamic acid decarboxylase (GAD₆₇ mRNA) in the striatum of five patients with Alzheimer's disease (AD) and nine matched control subjects. We found a 51–57% increase in the optical density of hybridization signal in the caudate nucleus and putamen, corresponding to a 30–42% increase in the number of neurons expressing a detectable amount of GAD₆₇ mRNA. By contrast, no alteration was observed in the ventral striatum. The expression of GAD₆₇ mRNA per neuron was similar in AD and control subjects both in the dorsal and ventral striatum. Taken together, our data indicate that, in AD, GABAergic neurotransmission is increased in the dorsal striatum but not in the ventral striatum. We suggest that this increased GABAergic neurotransmission may explain extrapyramidal signs often observed in AD.     

Heteromers of Glutamate Decarboxylase Isoforms Occur in Rat Cerebellum

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD₆₅ and GAD₆₇). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD₆₅ and GAD₆₇, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₇ was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₅ remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD₆₅ and GAD₆₇ even after extensive washing. These results are consistent with the presence of heteromultimers of GAD₆₅ and GAD₆₇ in cerebellum in addition to homomers of each form.     

Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

Molecular Cloning of Two Additional Members of the Neural Visinin-Like Ca2+-Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin-like Ca²⁺-binding protein (NVP), which we designate NVP-1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP-1 cDNA probe under low-stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP-2 and NVP-3, have been isolated. The deduced amino acid sequences of NVP-2 and NVP-3 are 89.0% and 68.6% identical to that of NVP-1, respectively, and contain consensus sequences for EF-hand Ca²⁺-binding sites. Northern blot analysis shows that NVP-1, NVP-2, and NVP-3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP-2 and NVP-3 are additional members of the NVP gene family.     

Streptococcus pneumoniae type 3 encodes a protein highly similar to the human glutamate decarboxylase (GAD65)

Abstract A 2.5-kb Sca I fragment of the type 3 pneumococcal strain 406 DNA containing a 1425-nucleotide open reading frame ( gadA ) and encoding a 475-amino acid protein ( M _rmr 54427) was characterised. The gene gadA was expressed in Salmonella typhimurium . Pulsed-field gel electrophoresis and Southern blotting analysis of DNAs prepared from several pneumococcal serotypes showed that only those clinical isolates belonging to serotype 3 harbour the gadA gene. Sequence comparison of GadA with proteins included in the data banks revealed the highest similarity with human glutamate decarboxylase (GAD₆₅) (59% similarity, 28% identity). Auto-antibodies to GAD₆₅ have been associated with the onset of insulin-dependent diabetes mellitus. Interestingly, several epitopes of GAD₆₅ that have been identified as immunodominant are particularly well conserved in the pneumococcal GadA.     

The Effect of pH on Glycolysis and Phosphofructokinase Activity in Cultured Cells and Synaptosomes

Abstract: Several G_i-linked neurotransmitter receptors, including dopamine D₂ receptors, act synergistically with Ca²⁺-mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor-dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D₂-receptor cDNA, CHO(D₂). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA-specific phospholipase A₂ (cPLA₂) were synthesized and added to cultures of CHO(D₂) cells. Incubation with antisense oligodeoxynucleotides inhibited D₂ receptor-dependent release of AA but had no effect on D₂-receptor binding or D₂ inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D₂ receptor-dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane-bound, non-AA-specific PLA₂ (such as p -bromophenacyl bromide). cPLA₂ is expressed in brain tissue. The results, showing that cPLA₂ participates in receptor-dependent potentiation of AA release in CHO(D₂) cells, suggest that this phospholipase may serve a similar signaling function in brain.     

Cloning, Characterization, and Distribution of a μ-Opioid Receptor in Rat Brain

Abstract: We report the isolation and characterization of a rat cDNA clone encoding a μ-opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ-and K -opioid receptors. Transient expression of the receptor in COS cells revealed high-affinity binding of μ-selective opioid antagonists and agonists, with a K _D for naloxone ∼1.5 n M , and for [D-Ala², N -Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) and morphine at the high-affinity site of 2–4 n M , confirming a μ-opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ-opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ-opioid receptor.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

Molecular Cloning, Expression, and Chromosomal Localization of a Human Brain-Specific Na+-Dependent Inorganic Phosphate Cotransporter

Abstract: We describe the molecular cloning of a cDNA encoding a human brain Na⁺-dependent inorganic phosphate (P_i) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na⁺-dependent inorganic P_i cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS-1 cells results in Na⁺-dependent P_i uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron-enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia-enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late-onset familial Alzheimer's disease locus.     

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5-HT₆ serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT₆ cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT₆ receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT₆ amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT₆ receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5-HT_1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT₆ receptor also reveals an Rsa I restriction fragment length polymorphism within the coding region.     

Dominant Expression of Rat Prostanoid DP Receptor mRNA in Leptomeninges, Inner Segments of Photoreceptor Cells, Iris Epithelium, and Ciliary Processes

Abstract: Prostaglandin (PG) D₂ is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD₂ among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD₂.     

Reaction of Muscimol with 4-Aminobutyrate Aminotransferase

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA-T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, K _m (app) = 1.92 ± 0.24 m M , specific activity = 7.33 ± 0.27 μmol/min/mg ( k _cat= 13.7s⁻¹), and for muscimol, K _m (app) = 1.27 ± 0.15 m M , specific activity = 0.101 ± 0.009 μmol/min/mg ( k _cat= 0.19s⁻¹). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first-order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K = 1.1 ±0.18 m M and k _max= 0.065 ± 0.004 s⁻¹ (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA-T.     

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABA_A) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABA_A receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [³H]muscimol and [³H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.