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

Abstract— Severe vitamin B₆ deficiency induced in pregnant rats during the last 2 weeks of gestation resulted in a reduction of brain weight in the new born rat. This indicates that the foetus was affected in utero . However, no significant changes were observed in other measured parameters in brains of the neonates at birth. Subjecting these neonates to vitamin B6 deficiency during lactation severely retarded the development of their body and brain weights. There is evidence to suggest that B₆ deficiency also leads to increased levels of glutamic acid decarboxylase apoenzyme, although the in vivo activity of the enzyme appears to be reduced as a result of marked reduction in coenzyme saturation. The level of γ-aminobutyric acid transaminase apoenzyme was reduced. Its coenzyme saturation was also reduced, but the level of reduction was less than with the decarboxylase. The progressive increase in whole brain γ-aminobutyric acid level was also retarded by the deficiency. Five days after administration of pyridoxine hydrochloride to 2-week-old deficient neonates, whole brain γ-aminobutyric acid levels and the activities of whole brain glutamic acid decarboxylase and γ-aminobutyric acid transaminase were almost restored to normal. However, brain and body weight showed a slow recovery during the same period. It was found that in the recovering neonates both enzymes follow changes in age rather than changes in brain weight.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.     

Retina Maturation Following Administration of Thyroxine in Developing Rats: Effects on Polyamine Metabolism and Glutamate Decarboxylase

Abstract: The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and γ-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9–12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S -Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.     

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.     

Effects of cell signaling on the development of GABA receptors in chick retina neurons

R-cognin, a cell recognition molecule, and insulin are known to play significant roles in GABAergic differentiation in the developing chick retina. In the present study, the effects of insulin and R-cognin on post-synaptic (GABAceptive) differentiation were investigated. In ovo binding of [³H]GABA and [³H]flunitrazepam ([³H]Flu) to the GABA and benzodiazepine (BZD) receptors, respectively, remained at low levels during early embryogenesis but increased sharply from mid-embryogenesis through hatching, increases which also occur in cultured neurons from early-embryonic (E7) and mid-embryonic (E11) chick retina. E7 neurons respond to insulin treatment (100 ng/ml) with increased [³H]Flu binding but no change in [³H]GABA binding. Cognin antibody (10 g/ml) treatment of E7 neurons caused no significant inhibition of the developmental increases in binding of either radioligand. Insulin in E11 cultures led to greater developmental increases in binding sites for both radioligands, but exposure to cognin antibody was without significant effect. These data, along with previous studies, indicate that GABAergic differentiation in developing chick retina is regulated, in part, by insulin and cognin-mediated cell signaling. Insulin also regulates post-synaptic (GABAceptive) differentiation whereas cognin-mediated interactions are relatively insignificant.Abbreviations BZD benzodiazepine - ChAT choline acetyltransferase - Flu flunitrazepam - GABA -aminobutyric acid - GAD glutamate decarboxylase (glutamic acid decarboxylase)     

Effect of 5''-deoxy-5''-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts.

The effect of 5'-deoxy-5'-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.     

The Inactivation of γ-Aminobutyric Acid Transaminase in Dissociated Neuronal Cultures from Spinal Cord

Abstract: It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter γ-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10⁻⁶ M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.     

Expression of Neuronal Specificities in "Transdifferentiating" Cultures of Neural Retina

Cells dissociated from the neural retina of embryonic chick differentiate into lens and pigment cells, when cultured in vitro. Using 3.5-day-old and 8.5-day-old chick embryos, we examined whether neuronal specificities would be expressed in such transdifferentiating cultures of neural retinal cells. The synthesis of acetylcholine and γ-aminobutyric acid (GABA) and the activity of choline acetyl transferase (CAT) was searched for in these cultures. The synthesis of an appreciable amount of these two putative neurotransmitters was detected in cultures of 3.5-day-old embryonic retinas by about 15 days. The activity of CAT was maximum in 7-day cultures of the 3.5-day-old materials and in 2-day cultures of the 8.5-day-old materials, and then decreased. Concomitant with the decrease of CAT-activity, δ-crystallin became detectable and increased thereafter. CAT-activity changed in parallel with the increase in the number of small neuroblast-like cells in cultures. The results demonstrate that the neuronal specificity identified by the appearance of acetylcholine and GABA and of the enzyme for the synthesis of acetylcholine is expressed in the early period of transdifferentiating cultures, which would later differentiate into lens and pigment cells. The possible mechanisms of the transition from neuronal to non-neuroretinal specificities of the transdifferentiating cultures are discussed.     

Anandamide, a Brain Endogenous Compound, Interacts Specifically with Cannabinoid Receptors and Inhibits Adenylate Cyclase

Abstract: We investigated the role of polyamines and their regulatory enzyme ornithine decarboxylase in N -Methyl-D-aspartate-induced excitotoxicity in embryonic chick retina. N -Methyl-D-aspartate (200 μM) produced an early increase in ornithine decarboxylase activity, putrescine concentration, and Ca²+ entry, leading to selective neuronal death by 30 min. This response was attenuated by the ornithine decarboxylase inhibitor α-difluoromethylornithine and the N -methyl-D-aspartate receptor antagonist 5-aminophosphonovaleric acid. Exogenous putrescine increased intracellular putrescine and spermine levels and reversed neuroprotection by α-difluoromethylornithine, but not by 5-aminophosphonovaleric acid. N -Methyl-D-aspartate-receptor stimulation of putrescine/polyamine synthesis mediates abnormal Ca²+ entry and acute excitotoxic neuronal death. Postreceptor inhibition of the ornithine decar-boxylase/polyamine cascade by α-difluoromethylornithine may provide neuroprotection against N -methyl-D-aspartate-induced excitotoxicity.     

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.     

Anaerobic Amino Acid Metabolism

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, -aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.     

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.     

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.     

Metabolic routes of GABA formation in chick embryo brain

Sobue and Nakajima (1978) reported that GABA formation from putrescine is significant in chick embryo brain between days 6 and 8 of incubation. They attributed an important functional role to the putrescine-derived GABA. We found that depletion of putrescine and spermidine in chick embryos by inhibition of ornithine decarboxylase activity did not decrease the in vivo rate of GABA formation, showing that putrescine is, from a quantitative point of view, a negligible source for GABA in chick embryo brain. The changes of brain GABA levels obtained after administration of glutamate decarboxylase inhibitors and in vitro determinations of glutamate decarboxylase activity were compatible with the assumption that GABA is mainly formed by decarboxylation of l-glutamate, even during early brain development. Participation of the NAD⁺-dependent, aerobic transformation of glutamate into GABA (Seiler and Wagner, 1976) in the overall GABA production of chick embryo brain could, however, not be excluded.     

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.     

REGIONAL DISTRIBUTION OF AMINO ACIDS IN HUMAN BRAIN OBTAINED AT AUTOPSY

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in 12 different regions of each of five human brains. Specimens were obtained at autopsy from patients who died suddenly without previous brain disease. These data may serve for later comparison with contents of amino compounds in similar regions of the brains of patients dying with various neurological or psychiatric disorders.
There were marked and consistent differences in the regional distribution of the following eight compounds: γ-aminobutyric acid, homocarnosine, glutamic acid, aspartic acid, taurine, cystathionine, glycerophosphoethanolamine, and phosphoethanolamine. These differences suggest that some of these compounds may have special physiological roles, including the possible mediation of synaptic transmission.
Human brain contains two previously unreported compounds, the mixed disulphide of cysteine and glutathione and α-(γ-aminobutyryl)-lysine. The latter dipeptide occurs in much higher concentrations in human brain than in the brains of lower mammals.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

Content of free amino acids and their exosmose from maize kernels in relation to cold resistance

Maize caryopses were exposed in moist, sterile sand to temperatures of 24°, 10° and 6°. The amino acid content of embryo, endosperm and pericarp tissues of kernels and amino acid occurence in sand eluates was analysed by paper chromatography. The lower the temperature to which the seeds were exposed and the greater the delay in start of germination, the greater were the amounts of amino acids exosmosed from the kernel to the surrounding media. The exudation of aspartic acid, proline, alanine, γ-aminobutyric and glutamic acids was especially high. The time-course of amino acid content in various kernel parts proves that cold does not check the enzymic hydrolysis of reserve nitrogen compounds. There is, however, an inhibition of translocation and use of amino acids in the embryo. Therefore, amino acids diffuse out from the caryopse into the media. Although mainly the peripheral parts of endosperm take part in exosmose, participation of the embryo in the exudation of certain amino acids (proline) can be supposed as well. In cold resistance of emerging maize kernels the remarkable interaction of microorganisms is causally related to the high amino acid exosmose found in these experiments. The mechanism of amino acid exosmose and its relationship to cold resistance are discussed in detail.