STUDIES ON THE RELATIONSHIP BETWEEN GABA SYNTHESIS AND PROTEIN SYNTHESIS IN BRAIN

Abstract— The synthesis of γ-aminobutyric acid (GABA) in mouse brain was decreased by treatment of the animals with pyridoxal phosphate- γ-glutamylhydrazone, an inhibitor of glutamate decarboxylase in vivo. Under these experimental conditions the following parameters were studied: (1) the incorporation of labeled leucine in vivo , into protein of brain subcellular fractions; (2) the brain polysome profile; (3) the incorporation of labeled leucine into protein in vitro , in ribosomal preparations isolated from brain tissue. In other experiments, GABA synthesis was also decreased in brain cortex slices by preincubation with aminooxyacetic acid. The incorporation of [³H]leucine or [¹⁴C]leucine into protein in these slices was studied, and samples from the proteins were subjected to acrylamide-sodium dodecylsulfate gel electrophoresis. Radioactivity was counted in slices of the gel. The results of the experiments in vivo and in vitro indicate that the previously reported decrease of protein synthesis induced by an inhibition of GABA synthesis affects proteins of all subcellular fractions and all populations of protein as separated by gel electrophoresis. The polysome profile from brains of mice with decreased GABA synthesis was similar to that of control mice. This result differs from that found when brain protein synthesis is inhibited by dopamine and serotonin.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

THE EFFECTS OF SOME NEWER ANAESTHETICS ON THE IN VITRO ACTIVITY OF GLUTAMATE DECARBOXYLASE AND GABA TRANSAMINASE IN CRUDE BRAIN EXTRACTS AND ON THE LEVELS OF AMINO ACIDS IN VIVO

—The effects of several anaesthetic and hypnotic compounds with well-defined excitatory side-effects on glutamate decarboxylase and γ-aminobutyric acid transaminase activity have been examined. The dissociative anaesthetics ketamine and γ-hydroxybutyric acid produced competitive inhibition of glutamate decarboxylase with respect to glutamate at concentrations which had no effect on GABA transaminase activity. The inhibitor constant (K_i) values were, ketamine: 13.3 mm , γ-hydroxybutyric acid; 8.8 mm . The steroid anaesthetic alphaxalone was also a potent competitive inhibitor of glutamate decarboxylase K_i= 4.1 mm ). Pentobarbitone, thiopentone and methohexitone non-competitively inhibited both glutamate decarboxylase and GABA-transaminase but only at high concentration (> 20 mm ). None of the drugs tested produced any significant change in brain GABA or glutamate levels following the injection of an hypnotic or anaesthetic dose. It is proposed that an alteration in the rate of GABA synthesis as a result of the inhibition of glutamate decarboxylase could explain the convulsive properties of the dissociative anaesthetics when given at high doses.     

EFFECTS OF UNDER NUTRITION AND PROTEIN DEFICIENCY ON GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN RAT BRAIN

Abstract— Studies were made on the effects of undernutrition at different ages during the neonatal period and of the comparative effects of postweaning protein and calorie deficiencies in neonatally undernourished or normally reared animals. Neonatal undernutrition resulted in deficits in body wt, brain wt and the activities of brain glutamate dehydrogenase and glutamate decarboxylase. Percentage deficits in brain wt were maximum in the first week of life but those in brain enzymes were greater in the second week. Rehabilitation of neonatally undernourished animals reversed the deficits in brain wt and brain enzymes. Post-weaning protein deficiency produced similar deficits in brain enzymes in both neonatally undernourished and normally reared animals. With post-weaning undernutrition, however, these deficits were found only in animals subjected to neonatal undernutrition as well.     

STUDIES ON THE REGULATION OF GABA SYNTHESIS: SUBSTRATE-PROMOTED DISSOCIATION OF PYRIDOXAL-5''-PHOSPHATE FROM GAD

The association between glutamate decarboxylase (GAD) and its cofactor, pyridoxal-5′-phos-phate (pyridoxal-P), was studied using 20,0000 supernatant of rat brain. In this preparation GAD required added pyridoxal-P to maintain a linear reaction rate beyond 5 min of incubation. Following exhaustive dialysis the enzyme was more than 83% saturated with cofactor indicating that the cofactor was tightly bound to the enzyme. When incubations were performed in the presence of glutamate and without added pyridoxal-P there was a progressive inactivation of the enzyme which was dependent on the glutamate concentration. This lost activity was almost completely recovered by addition of pyridoxal-P to the dialyzed glutamate-inactivated enzyme. The results suggest that glutamate inactivates GAD by promoting the dissociation of pyridoxal-P from the enzyme thereby producing inactive apoen-zyme which can be reactivated by combining with available pyridoxal-P. This interpretation is supported by the finding that progress curves for the reaction were accurately described over a 30 min incubation period and 10-fold glutamate concentration range by an integrated rate equation which takes the glutamate-promoted dissociation of cofactor into account. The progressive inactivation could not be attributed to denaturation of the enzyme, impurities in the substrate, effects of pH, depletion of substrate, protein concentration, sulfhydryl reagents or product inhibition. The results presented here also show that certain precautions must be adopted to accurately measure GAD activity in the absence of added pyridoxal-P as has been widely done in studies of drug action. Specifically, measurements must be made at short times of incubation and low concentrations of glutamate to minimize the glutamate-promoted inactivation of the enzyme.     

THE EFFECT OF β,β''-IMINODIPROPIONITRILE AND ANAESTHESIA ON SOME ADENINE NUCLEOTIDES OF RAT BRAIN AND RETINA

—A significant increase in the retinal ATP content of anaesthetised rats was found 6 days after administration of β, β′-iminodipropionitrile (IDPN). With the development of retinal dystrophy variable ATP levels were observed from the 8th to the 12th day and low values were recorded on the 17th and 21st days. At 8 days after IDPN administration the ATP content of anaesthetised rat brain was significantly increased with slight decreases in ADP and AMP levels. The differences in the level of these adenine nucleotides in unanaesthetised and anaesthetised rat brain were not significant before or after IDPN administration. These results were related to previous experiments on the action of IDPN on the electroretinal response and the later development of a retinal microangiopathy. It was suggested that IDPN has a primary neurotoxic effect followed by the development of vascular morphological changes.     

DIFFERENTIAL EFFECTS OF AGONAL STATUS ON MEASUREMENTS OF GABA AND GLUTAMATE DECARBOXYLASE IN HUMAN POST-MORTEM BRAIN TISSUE FROM CONTROL AND HUNTINGTON''S CHOREA SUBJECTS

Abstract— GABA and its biosynthetic enzyme glutamic acid decarboxylase (GAD) remained remarkably stable for many hours after death in both human putamen obtained at autopsy and in mouse brain stored under conditions simulating the routine handling of human cadavers. GAD activity was profoundly influenced by agonal status in control but not in choreic subjects. Conversely, GABA concentrations were unaffected by the agonal status but showed a significant age-related decline. GAD activity and GABA concentrations were positively correlated in sudden death control cases but not in control cases suffering a protracted terminal illness or in choreic subjects. In choreic putamen there was an approximate 50% reduction in GABA concentration and GAD activity (correcting for agonal status) consistent with the hypothesis that striatal GABA-containing neurones degenerate in this disease. Since GABA concentrations are unaffected by agonal factors they may provide a reliable marker for the integrity of GABA systems provided that control and pathological groups are matched for age and delay in post-mortem sampling.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.     

REGIONAL DISTRIBUTION OF GLUTAMATE DECARBOXYLASE AND GABA WITHIN THE AMYGDALOID COMPLEX AND STRIA TERMINALIS SYSTEM OF THE RAT

The distribution of glutamate decarboxylase (GAD) and δ-amino butyric acid have been studied in the amygdaloid complex and in the stria terminalis system of the rat. The central and medial nuclei of the amygdala had significantly higher activities of GAD than the lateral olfactory tract nucleus, anterior amygdala, anterior lateral nucleus, posterior lateral nucleus, cortical nucleus, basomedial nucleus, basolateral nucleus, and pyriform cortex. The enzyme activity was about two and a half times higher in the central and medial nuclei than in the pyriform cortex. GABA was also significantly more concentratcd in these nuclei than in the pyriform cortex although this was not true for four other amino acids studied–glutamic acid, aspartic acid, taurine and glycine. GAD activity was also measured in the stria terminalis (the major afferent and efferent pathway of the amygdala) and in its bed nucleus. The enzyme activity was higher in the stria terminalis than in four other fibre tracts studied–the optic tract, anterior commissure, corpus callosum, and fimbria. GAD activity was exceptionally high in the bed nucleus of the stria terminalis particularly in its ventral part. The significance of the results are discussed in terms of what is known about the evolution and anatomy of the amygdala.     

CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES

The binding of glutamate decarboxylase (GAD), to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca²⁺ and Mg²⁺ concentrations was studied. Phosphatidylcho-line-phosphatidylserine (4:1) liposomes are capable of binding GAD in a Ca²⁺-dependent manner. The per cent of GAD bound increased from 5 to 65°., in a sigmoid shape with Ca²⁺ concentrations in the 0.2-4 mm range. Mg²⁺ also induces GAD binding but is less effective than Ca²⁺ The Ca²⁺ -dependent binding of GAD is not the result of unspecific association of protein, since Ca²⁺ did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore, the relative specific activity (^o_o enzyme activity/% protein) of GAD associated to liposomes increases 4-fold from 0 to 2 mm Ca²⁺. The per cent of GAD bound attains a plateau at a ratio phospholipid/protein of about 1.5. and decreases when the pH increases from 6.5 or 6.8 to 7 or 7.25. Na⁺ or K⁺ at a 100mm concentration also induce binding of GAD to liposomes. Phosphatidylcholine liposomes (without phosphatidylserine) practically did not bind GAD at any Ca²⁺ concentration. The Ca²⁺-dependent association of GAD to phosphatidylcholine-phosphatidylserine liposomes is very similar to that previously reported using brain membranes, and it correlates also well with the reported Ca²⁺-dependent aggregation of phosphatidylserine molecules in phospholipid membranes of similar composition. It is concluded that phosphatidylserine is probably involved in the Ca²⁺-dependent binding of GAD to brain membranes. Phospholipid vesicles seem to be a useful experimental model for studying the mechanisms of this GAD association to membranes and the possible physiological implications of the GAD-Ca²⁺-membrane interaction regarding the release of newly synthesized GABA from nerve endings.     

EFFECTS OF DIFFERENT LEVELS OF DIETARY PROTEIN ON BRAIN GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN YOUNG ALBINO RATS

Abstract— Studies were carried out to identify the minimum levels of protein (casein) needed in the diet in order to prevent or reverse the deficits in brain enzymes previously found with protein deficiency. Groups of weanling albino rats were fed diets containing variable amounts of protein (5, 8, 10, 15 or 20 per cent in experiment I, and 5, 6, 7, 8 or 20 per cent in experiment II) for 5 or 10 weeks. Deficits in brain wt and brain glutamate dehydrogenase and decarboxylase were found to be prevented by a diet containing 8 per cent or more of protein, although for optimum growth 15 per cent protein in the diet was found to be necessary. Groups of rats were fed a 5 or 20% protein diet for 10 weeks after which the 5% protein animals were either continued on the diet for another 10 weeks or changed to one containing 8, 10, 15 or 20% protein. The brain enzyme deficits found with the 5% protein diet were found to be fully reversed by feeding a 10% protein diet during rehabilitation.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

铝与钙调蛋白相互作用及其作用位点的研究

用500MHzNMR研究了铝与钙调蛋白的相互作用,主要研究了铝对钙调蛋白中芳香氨基酸残基(Tyr,His,Phe)构象变化的影响。实验结果表明,铝在钙饱和的钙调蛋白上存在着特异性的结合位点,结合位点数目至少为两个,第一结合位点可能位于钙调蛋白的N端结构域,第二结合位点靠近Ca~(2+)的Ⅲ结合域。Al~(3+)结合引起脱钙的钙调蛋白的构象变化不同于与Ca~(3+)结合引起的构象变化。Al~(3+)在CaM上的结合位点与Ca~(2+)的并不相同。柠檬酸等有机酸对铝的毒性有保护作用,这种保护作用是由于柠檬酸分子对铝的络合。     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INHIBITION STUDIES WITH N-(5′-PHOSPHOPYRIDOXYL) AMINO ACIDS1

Abstract— Seven N-(5′-phosphopyridoxyl) amino acids, reduced analogs of the glutamate-pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul-finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non-linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

褪黑素对谷氨酸致痫大鼠海马内谷氨酸、GABA及其受体水平的影响

目的探讨褪黑素(melatonin,MT)对谷氨酸(glutamate,Glu)致痫大鼠海马内Glu及GluR2、γ-氨基丁酸(γ-aminobutyric-acid,GABA)及其受体GABRA1水平的影响,进而研究褪黑素的抑痫作用机制。方法随机将健康SD雄性大鼠40只分为A、B、C、D组,每组10只。A组:生理盐水组;B组:MT Glu组;C组:Glu致痫组;D组:Luzidole MT Glu组。观察并记录行为学变化,采用免疫组化法进行Glu、GluR2、GABA和GABRA1免疫组化染色和图像分析。结果行为学观察结果显示,C组和D组大鼠均有不同程度的癫痫发作,B组大鼠癫痫发作不明显,A组无发作;免疫组化结果显示,C组和D组海马内CA1-CA3区和齿状回Glu阳性反应较A组增强(P<0.05),GluR2、GABA和GABRA1均较A组减弱(P<0.05),B组Glu较C组和D组阳性反应有显著性减弱(P<0.05),GluR2、GABA和GABRA1阳性反应均较C组和D组有显著性增强(P<0.05),而B组与A组无明显差异性。结论MT通过增加GABA及其受体GABRA1和GluR2的作用和抑制Glu作用对Glu致痫大鼠癫痫发作发挥抑制作用。