SOME EFFECTS OF DIETARY VITAMIN B6 DEFICIENCY AND 4-DEOXYPYRIDOXINE ON γ-AMINOBUTYRIC ACID METABOLISM IN RAT BRAIN

Abstract— Investigations of the respective effects of dietary vitamin B₆ deficiency and 4-deoxypyridoxine (a vitamin B₆ antagonist) on GABA metabolism in rat brain have been carried out. No convulsions were observed in rats subjected to either treatment. GABA levels were lowered by both treatments, the greatest diminutions being found with the dietary deficiency. Glutamic acid decarboxylase activity was reduced under both conditions, but the loss of activity in the B₆ deficiency experiments could be attributed to cofactor depletion, whereas in the deoxypyridoxine experiments the loss of activity appears to be due to lower levels of available apoenzyme. The activity of GABA-transaminase was not affected by deoxypyridoxine treatment and only moderately reduced in the B.5 deficient animals.     

Metabolism and Brain Uptake of γ-Aminobutyric Acid in Galactosamine-Induced Hepatic Encephalopathy in Rats

Abstract: Kinetic studies of [³H]γ-aminobutyric acid ([³H]GABA) after an intravenous injection were performed in normal rats and in rats with severe degree of hepatic encephalopathy due to fulminant hepatic failure induced by galactosamine. Moreover, plasma and brain GABA levels, and GABA and glutamic acid decarboxylase activity were studied in some brain areas. After intravenous injection, [3H]GABA disappeared very rapidly in the blood of normal rats, with a prompt increase of ³H metabolites. In comatose rats, a delayed disappearance of [3H]GABA.as parallelled by a lower amount of metabolites, indirectly indicating a peripheral decrease of GABA-transaminase activity. The amount of [³H]GABA in brain was lightly but constantly lower in comatose rats than in controls, indicating that the change in permeability of the blood-brain barrier in hepatic encephalopathy does not affect the [3H]GABA uptake of the brain. Furthermore, the assay of endogenous GABA in blood, whole brain, and brain areas did not show any significant difference in any of the two groups. The finding that glutamic acid decarboxylase activity in brain was reduced, together with the indirect evidence of a reduction in GABA-transaminase, may account for the steady state of GABA in hepatic encephalopathy. However, the reduction in glutamic acid decarboxylase activity is in favor of a functional derangement at the GABA-ergic nerve terminals in this pathological condition.     

ORNITHINE AND GLUTAMIC ACID DECARBOXYLASE ACTIVITIES IN THE DEVELOPING CHICK RETINA

Abstract— γ-Aminobutyric acid was found in 6-day-old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ-aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ-aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ-aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ-aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ-aminobutyric acid, catalyzed by glutamic acid decarboxylase.     

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.     

Characterization of Glutamic Acid Decarboxylase Activity in Cerebral Blood Vessels

Abstract: Glutamic acid decarboxylase activity associated with cerebral blood vessels appears to be part of a specific cerebrovascular system involving γ-aminobutyric acid. This activity was characterized kinetically and pharmacologically and compared with that in brain and several nonneuronal tissues. Formation of γ-aminobutyric acid from [¹⁴C]glutamate was measured in a soluble extract of pia-arachnoid blood vessels isolated from bovine brain. The vascular activity was like brain glutamate decarboxylase in that it required pyridoxal phosphate, was completely inhibited by aminooxyacetic acid, and had a similar affinity for glutamate. Cerebrovascular decarboxylase activity differed, however, from brain decarboxylase in that it was less sensitive to sulfhydryl reagents, was stimulated by 3-mercaptopropionic and cysteic acids, and was competitively inhibited by cysteine sulfinic acid. The glutamate decarboxylase activity of the cerebral vessels was similar to that in renal cortex and mesenteric blood vessels in its responses to sulfhydryl reagents and 3-mercaptopropionic acid. These findings are consistent with previous suggestions of a nonneuronal form of the enzyme and offer the possibility that synthesis of γ-aminobutyric acid in cerebral blood vessels can be manipulated independently from that in neuronal tissue.     

Distribution of Three Enzymes of γ-Aminobutyric Acid Metabolism in Monkey Retina

Abstract: The distributions of glutamate decarboxylase (EC 4.1.1.15), γ-aminobutyric acid transaminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC 1.2.1.24) were determined in monkey retina. The decarboxylase was almost restricted to the inner plexiform layer. The transaminase was also highest in this layer, but activities were 40% as high in the adjacent third of the inner nuclear layer and in the ganglion cell and fiber layers. Succinate semialdehyde dehydrogenase was distributed very differently. Although it also showed a peak of activity in the inner plexiform layer, there was a second equal peak in the photoreceptor inner segment layer and a smaller peak in the outer plexiform layer, regions where both γ-aminobutyric acid transaminase and glutamate decarboxylase were essentially absent.     

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 EFFECT OF 4-AMINO HEX-5-YNOIC ACID (γ-ACETYLENIC GABA, γ-ETHYNYL GABA) A CATALYTIC INHIBITOR OF GABA TRANSAMINASE, ON BRAIN GABA METABOLISM IN VIVO

Abstract— 4-Amino hex-5-ynoic acid (γ-acetylenic GABA, γ-ethynyl GABA, RM171.645), a catalytic inhibitor of GABA transaminase in vitro , induces a rapid, long-lasting dose-dependent decrease of GABA transaminase activity and, to a lesser extent, of glutamate decarboxylase activity in the brains of rats and mice when given by a peripheral route. The GABA concentration in whole brain increases up to 6-fold over control values. The action of γ-acetylenic GABA is relatively specific, as no in vivo inhibition of brain aspartate and alanine transaminase activities could be detected. Furthermore, the amount of radioactive drug bound to the protein fraction of brain homogenate is of the same order of magnitude as that of the GABA transaminase present, as calculated from total GABA transaminase activity, molecular weight and specific activity of the pure enzyme. γ-Acetylenic GABA illustrates the usefulness of a catalytic irreversible enzyme inhibitor in altering neurotransmitter metabolism in vivo .     

GABA-transaminase and glutamic acid decarboxylase changes in the brain of rats treated with pyrithiamine

Pyrithiamine, a thiamine phosphokinase inhibitor, was fed to rats on a thiamine-deficient diet, producing weight loss, ataxia and loss of righting reflex in 10 days. Some rats were then sacrificed; others were returned to a normal diet, to be sacrificed only when their weight had returned to pre-experimental levels. Rats were sacrificed for assay of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) activities in homogenates of eight brain regions or were perfused for -aminobutyric acid transaminase (GABA-T) histochemistry. GAD activity was significantly reduced in symptomatic rats in the thalamus > cerebellum > midbrain > pons/medulla. GABA-T staining was similarly reduced, with greatest losses in the thalamus > inferior colliculus > pons > medulla. ChAT activity was not significantly altered in any brain area. Following return to a normal diet, GAD activity was significantly recovered in all areas except the thalamus. GABA-T staining recovered, at least partially, in all areas affected.     

High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain

Abstract: Acute administration of vitamin B₆ to rats (10 mg/kg body weight) led to reduced urinary excretion of N ¹-methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of ¹⁴CO₂ from [ ring -2-¹⁴C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [³H]tryptophan, accompanied by a small increase in the rate of synthesis of 5-hydroxy-tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B₆ may explain the beneficial action of the vitamin in some cases of depressive illness.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.     

Tetanus Toxin Inhibition of K+ -Stimulated [3H]GABA Release from Developing Cell Cultures of the Rat Cerebellum

Abstract: The effect of tetanus toxin pretreatment on K⁺ -stimulated [³H]γ-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K⁺-evoked [³H]γ-aminobutyric acid release from 7- and 14-day-old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.     

Lipid Composition of the Brain in the Vitamin B12-Deficient Fruit Bat (Rousettus aegyptiacus) with Neurological Impairment

Abstract: The Egyptian fruit bat Rousettus aegyptiacus develops severe vitamin B₁₂ deficiency when fed a diet of fresh peeled fruit and water. In a group of bats fed this diet, B₁₂ concentrations in the serum and brain were low, and neurological impairment, evidenced by deficient or absent hindlimb groping or grasping ability and climbing difficulties, was manifest. Control bats fed the identical diet supplemented with B₁₂ showed no such changes. Fatty acid analysis of whole brain homogenates showed a higher level of 20:4 in the deficient group. Phosphatidylcholine showed a marginally higher percentage of 18:3. The total percentage of branched chain fatty acids of phos-phatidylethanolamine was four times higher in deficient brains, comprising 2% of the total. Sphingomyelin showed a slightly higher percentage of 15:0, and a significantly lower percentage of long chain fatty acids C-24 and longer ( p < 0.01). The compositions of nonhydroxy fatty acids in cerebroside were unchanged. Examination of phospholipids showed that 8.9 ± 0.4% of total phosphorus was present as sphingomyelin in deficient bats, compared with 11.9 ± 1.2% in control animals (p < 0.05). There were no statistically significant differences in the concentrations of total brain lipid, protein, phospholipid, glycosphingolipid, cholesterol and plasmalogen between B₁₂-deficient and control bats.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

The γ-Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ-Aminobutyrate-Elevating Agents: A Possible Role in Anticonvulsant Activity

Abstract: The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the γ-aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.     

家蚕体内因缺乏维生素B6而引起的若干代谢变动

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori 5龄幼虫,探讨了缺乏维生素B₆（VB₆）对蚕体氨基酸代谢、脂肪酸代谢以及转氨酶活力的影响。缺乏VB₆引起支链氨基酸分解代谢受阻,幼虫体液中大量积累亮氨酸、缬氨酸和异亮氨酸。同时因绢丝腺发育停滞,丝氨酸也在体液中积累。另一方面,缺乏VB₆幼虫体液中赖氨酸、脯氨酸、精氨酸、甲硫氨酸和谷氨酸含量减少,其中赖氨酸尤为突出。推测缺乏VB₆引起赖氨酸分解代谢亢进。结果还表明,缺乏VB₆幼虫体内脂肪酸代谢异常,谷丙转氨酶活力显著低下。     

Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS Vitamin B₁₂ deficiency arrests cell division in Euglena gracilis. B₁₂ starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B₁₂ deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.     

Distribution of Glycine, γ-Aminobutyric Acid, Glutamate Decarboxylase, and γ-Aminobutyric Acid Transaminase in Rabbit and Mudpuppy Retinas

Abstract: The distributions of glycine, γ-aminobutyric acid (GABA), glutamate decarboxylase (EC 4.1.1.15), and GABA transaminase (EC 2.6.1.19) were determined in rabbit and mudpuppy retinas. In both species, peak levels of the amino acids and the enzymes occurred in the inner plexiform layer. Glutamate decarboxylase was almost entirely confined to the inner plexiform layer. Determinations were also made of the GABA content of 107 individual putative amacrine cell somas from mudpuppy retina. About 30% of those somas were found to have high endogenous GABA levels.     

Studies on new intracellular proteases in various organs of rat. 2. Mode of limited proteolysis.

1. The mechanism of proteolysis of ornithine transaminase apoenzyme II by group-specific protease and the relation between the confirmations of ornithine transaminase and its susceptibility to group-specific protease were studied to elucidate the mode of action of the protease. 2. Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group-specific protease, while the holoenzyme was not. 3. Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group-specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme II was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively. 4. The initial step of intracellular proteins degradation is discussed on the basis of these results.