Kinya Kuriyama

Elevation of γ-aminobutyric acid in brain with amino-oxyacetic acid and susceptibility to convulsive seizures in mice: A quantitative re-evaluation

Abstract The time courses of changes in brain content of γ-aminobutyric acid (γABA) and sensitivity to electroconvulsive shock were studied in mice after administration of amino-oxyacetic acid (AOAA), an inhibitor of γABA-transaminase. Some correlative observations were made of ATP and glutamate contents and of susceptibility to seizures induced by strychnine, pentylenetetrazole (Metrazol), and picrotoxin. The increase in γABA concentrations as a function of time after AOAA administration (25 mg/kg) was biphasic. A plateau in γABA level was attained between 3 and 4 hr, and a subsequent secondary rise took place between 4 and 6 hr. Elevated values were still observed at the 24-hr period. No changes in ATP content were found after AOAA. There was a remarkable decrease in electroshock seizure incidence (75 mA stimulus) during the first 1·5 hr after AOAA. Subsequently, the susceptibility to seizures began to return to normal, attaining the control values at 6 hr, at which time the γABA content was maximal. Only during the first 1·5-hr period after AOAA was there a good correlation of decrease in seizure susceptibility with increase in γABA content. Similar findings were made on a more limited scale with regard to the parameters measured when chemical convulsants were employed. One of several possibilities suggested by the findings is that the increases in γABA levels and changes in seizure susceptibility after AOAA administration are completely unrelated. Another way to view the results is that soon after γABA-transaminase blockade with AOAA the increased levels of γABA may, indeed, decrease neuronal excitability; however, compensatory increases in excitatory influences or decreases in inhibitory influences other than γABA, or both, may take place with the consequent restoration of normal neuronal sensitivity even during the time when a gross elevation of γABA exists. Glutamic acid, a potentially important excitatory factor in the central nervous system, did not increase after AOAA.

An anion stimulated l-glutamic acid decarboxylase in non-neural tissues: Occurrence and subcellular localization in mouse kidney and developing chick embryo brain☆

Abstract The formation of γ-aminobutyric acid (GABA), a probable inhibitory transmitter in some areas of the vertebrate central nervous system, is catalyzed by an l -glutamic acid decarboxylase (GAD). This GAD is inhibited by anions and carbonyl-trapping agents and stimulated by the addition of pyridoxal phosphate. The α-decarboxylation of l -glutamic acid now has been shown to occur in kidney and several other non-neuronal tissues. However, the latter process requires high concentrations of anions for maximal activity. Chloride (Cl−) was the anion studied most thoroughly. In further contrast to brain, the kidney enzyme was activated by aminooxyacetic acid (AOAA), a typical carbonyl trapping agent, and unaffected by the addition of pyridoxal phosphate. Developing chick embryo brain showed Cl− activation of GAD activity early in development, and Cl− inhibition at later stages. The Cl− and AOAA-stimulated GAD persisted in developing chick embryo brain mitochondria and nuclei through periods of development in which the GAD activity of the synaptosomal fraction already was greatly inhibited both by Cl− and AOAA. In mouse kidney there was observed a chiefly mitochondrial localization of the Cl−-activated GAD. Pyruvate was the most effective organic anion tested in stimulating the kidney enzyme and was more effective than isomolar concentrations of Cl−. Additive stimulatory effects were observed in the presence of both pyruvate and Cl− in some concentrations. All of the evidence favors the hypothesis that at least two forms of GAD exist in animal tissues.

Some characteristics of binding of γ-aminobutyric acid and acetylcholine to a synaptic vesicle fraction from mouse brain

Abstract In a vesicle-rich subcellular fraction prepared from whole mouse brain the binding of γ-aminobutyric acid (GABA) and acetylcholine (ACh) appear to take place by different mechanisms and at different sites. There is an absolute requirement of Na + ions for the binding of GABA that cannot be replaced by other cations. The binding of ACh is markedly inhibited by Na + , so that there is no binding of ACh at concentrations of Na + which are even suboptimal for the binding of GABA. Pretreatment of the vesicles with acetone solutions destroyed the binding capacity for GABA, but increased that for ACh. The binding of ACh appears to resemble a cation exchange process; and several inorganic cations, ammonia, and a variety of ionizable amines and quaternary compounds were found capable of inhibiting the binding of ACh by the particles. The results suggest that one of the attributes of ACh binding sites on the vesicles is the presence of exposed highly ionized polyanionic constituents. Treatment with neuraminidase removed 68% of the total N -acetylneuraminic acid from the vesicles, decreased the binding capacity from ACh by 34%, and had no effect on GABA binding. Significant fractionation of the mixed vesicle population with regard to GABA content, glutamic decarboxylase activity, and binding capacity for GABA and ACh was not achieved by density gradient centrifugation or by electrophoretic techniques.

Association of the γ-aminobutyric acid system with a synaptic vesicle fraction from mouse brain

Summary γ-Aminobutyric acid (γABA), glutamic acid, and glutamic decarboxylase (GAD) have been found to be more tightly associated than lactic dehydrogenase, a cytoplasmic marker, with a centrifugally isolated and morphologically characterized mouse brain synaptic vesicle fraction. Some aspects of the morphology were discussed. γABA transaminase was virtually absent from the vesicles. The synaptic vesicles were found to accumulate 14 C-labeled γABA and glutamic acid by a Na + -dependent process at 0° C in the absence of added metabolites. The accumulation was not related to any process requiring metabolic generation of energy. After preloading with labeled γABA at 0°, incubation at 30° in the absence or presence of metabolites or inhibitors invariably resulted in a loss of γABA from the vesicles. Several experiments were performed with 14 C-labeled γABA that gave results consistent with the suggestion that there is a Na + -dependent binding of γABA to the membrane of the particles and that the binding sites function as carriers to mediate the movement of γABA in and out of the particles.

LOCALIZATION OF γ-AMINOBUTYRIC-α-OXOGLUTARIC ACID TRANSAMINASE IN MOUSE BRAIN*

The distribution of γ‐aminobutyric‐α‐oxoglutarate transaminase (GABA‐T) has been studied in mouse brain by histochemical visualization on sections, quantitative determination in grossly dissected brain regions, and in subcellular fractions prepared from whole brain homogenates. The results indicate that GABA‐T is predominantly a mitochondrial enzyme and that its concentration is particularly high in brain stem areas. The results are discussed in relation to the overall function of the GABA system in the CNS.

The γ-aminobutyric acid system in the developing chick embryo cerebellum

The time-sequence of the development of the components of the γ-aminobutyric acid (GABA) system in the chick embryo cerebellum was correlated with development as observed at both light and electron microscopic levels. Study also was made of the subcellular distribution pattern of the GABA system with the age of the embryo. Characteristic synapse-like structures were observed in the chick cerebellum as early as 11 days of incubation, the degree of synaptogenesis increasing greatly thereafter. The enzymes of the GABA system, l-glutamic decar☐ylase (GAD) and GABA-transaminase (GABA-T), began to increase much later in development than did the weight and protein content of the cerebellum. All of the data are consistent with the interpretation that the development of the whole GABA system temporally is better correlated with the development and increase in recognizable synaptic structures than with the accretion of the total mass of the cerebellum. The fractionation data at all stages showed GAD to be more highly concentrated in presynaptic endings than elsewhere and the GABA-T to be particularly high in the free mitochondria, which probably come from postsynaptic neuronal sites and from glial and endothelial cells. The results fit the suggestion that in the chick cerebellum GABA largely is formed at presynaptic sites and metabolized at postsynaptic sites onto which it is liberated, but definitive proof of this idea will only come when it will be possible to visualize GAD, GABA-T, and GABA at an ultrastructural level in specific sites of sections of the cerebellum.

L-glutamic acid decarboxylase: a new type in glial cells and human brain gliomas.

Human glial cells grown in culture and gliomas and white matter contain an l-glutamic acid decarboxylase which is stimulated markedly by carbonyl-trapping agents. In contrast, L-glutamic acid decarboxylase activity of human cerebral gray matter is strongly inhibited by carbonyl-trapping agents. These results suggest a glial localization of the new type of l-glutamic acid decarboxylase.

Uptake of γ-aminobutyric acid by mitochondrial and synaptosomal fractions from mouse brain

Summary The accumulation of γ-amino[ 14 C]butyric acid ([ 14 C]GABA) by purified nerve ending particles (synaptosomes) and mitochondria of mouse brain was investigated in the presence of aminooxyacetic acid, which prevents metabolic degradation of GABA. The accumulation of GABA by synaptosomes and brain mitochondria was Na + dependent and involved both energy independent and dependent accumulation processes. Pyruvate and glucose stimulated synaptosomal GABA accumulation at 30°C and that of mitochondria was stimulated by pyruvate and not by glucose. 2,4-Dinitrophenol, ouabain and malonate inhibited energy dependent accumulation of GABA by synaptosomes and brain mitochondria, and iodoacetate only affected the glucose stimulated synaptosomal uptake of GABA at 30°C. Results were presented which demonstrate a marked time dependent loss of uptake capacity by the subcellular fractions. A rapid procedure for obtaining a functionally superior mitochondrial fraction was described.

Electrophoretic mobilities of brain subcellular particles and binding of γ-aminobutyric acid, acetylcholine, norepinephrine, and 5-hydroxytryptamine

Electrophoretic mobilities of brain synaptosomes and synaptic vesicles and of brain, liver, and kidney mitochondria were determined in a continuous sucrose density gradient. All of the above particles were negatively charged with isoelectric points in the neighborhood of 4. γ-Aminobutyric acid was bound well by the vesicles, synaptosomes, and mitochondria from brain, but not at all by mitochondria from liver or kidney. Acetylcholine was bound to vesicles and synaptosomes but only slightly to the mitochondrial preparations. The binding of 5-hydroxytryptamine to the vesicles, and synaptosomes was much higher than to any of the mitochondrial preparations; while the binding of norepinephrine was much more evenly distributed among the particles. The results suggest that the binding capacities of the particles tested for the above 4 substances are not directly related to the net negative charge, and that the net negative charge on the particles cannot be attributable to that bound form of N-acetylneuraminic acid that can be removed by the action of neuraminidase.

Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain

Summary The acid mucopolysaccharide (AMPS) of mouse brain consist of predominantly of the chondroitin sulfate and also contains hyaluronic acid and small amounts of unidentified components. The amount of AMPS per g of delipidized dry materials was found to be highest in the synaptic vesicle fraction, which contained about twice as much as the whole brain particulate fraction. The content in the mitochondrial fraction was very low on lipid-free dry weight basis. About 34% of the total amount of AMPS in whole brain is present in a soluble state, either as free AMPS or as AMPS-protein complex.