Chronic metabolic effects of ammonia in mouse brain.

Chronic ammonia toxicity in experimental mice was induced by exposing them for 2 and 5 days to 5 % (v/v) ammonia solution. The enzymes concerned with glutamate metabolism (aspartate-, alanine- and tyrosine aminotransferases, glutamate dehydrogenase and glutamine synthetase) and (Na+ + K+)-ATPase were estimated in the three regions of brain (cerebellum, cerebral cortex and brain stem) and in liver. Glutamate, aspartate, alanine, glutamine and GABA, RNA and protein were also estimated in the three regions of brain and liver. A significant rise in the activity of (Na+ + K+)-ATPase in all the three regions of brain along with a fall in the activity of alanine aminotransferase was noticed. Changes in the activities of other enzymes were also observed. A significant increase in alanine and a decrease in glutamic acid was observed while no change was observed in the content of other amino acids belonging to the glutamate family. As a result of this, changes in the ratios of glutamate/glutamine and glutamate + aspartate/GABA was observed. The results indicated that the brain was in a state of more depression and less of excitation. Under these conditions the liver tissue was showing a profound rise in the activity of the enzymes of glutamate metabolism. The results are further discussed.     

Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain.

Acute and chronic ammonia toxicity was produced in the mice by intraperitoneal injection of ammonium chloride (200 mg/kg) and by exposure of mice to ammonia vapours (5% v/v) continuously for 2 days and 5 days respectively. The ammonia content was elevated in the cerebellum, cerebral cortex and brain stem and in liver. In acute ammonia intoxication there was a decrease in the monoamine oxidase (MAO) activity in all the three regions of brain. In chronic ammonia toxicity (2 days of exposure) a significant increase in the activity of MAO was observed in the cerebral cortex while in cerebellum and brain stem there was a significant decrease. In cerebral cortex and cerebellum there was a rise in the activity of MAO as a result of exposure to ammonia vapours for 5 days. A significant decrease was observed in the activity of glutamate decarboxylase (GAD) in all the three regions of the brain both in acute and chronic ammonia toxicity (2 days). There was a decrease in the activity of this enzyme only in the cerebral cortex in the animals exposed to ammonia for 5 days. The activity of GABA-aminotransferase (GABA-T) showed a significant rise in cerebellum and a fall in the brain stem in acute ammonia toxicity. In chronic ammonia toxicity GABA-T showed a rise in all the three regions of brain. Chronic ammonia toxicity produced a significant decrease in the content of glutamate in all the three regions without a significant change in the content of aspartate. GABA and glutamine. The content of alanine increased in all the three regions of brain under these experimental conditions. The ratio of glutamate + aspartate/GABA and glutamate/glutamine showed a decrease in all the three regions as a result of ammonia toxicity.     

Incorporation of 14C from glucose into amino acids in brain in vitro in the presence of organo- and inorganic lead and pyridoxal phosphate

The incorporation of label from U14C glucose into glutamic acid, glutamine and GABA remained unaltered with the presence of lead acetate in the medium whereas tetraethyl lead (TEL) affected the incorporation in a characteristic manner in different regions of brain. Glucose uptake however was not influenced by TEL. Pyridoxal phosphate was found to reverse the effect of TEL on the incorporation especially in cerebellum and brainstem but with little effect in cerebral cortex. These findings suggest that the alterations in the GABA metabolism in TEL toxicity could be restored to some extent by pyridoxine in discrete brain areas.     

Bilobalide prevents reduction of gamma-aminobutyric acid levels and glutamic acid decarboxylase activity induced by 4-O-methylpyridoxine in mouse hippocampus

We previously reported that bilobalide, a constituent of Ginkgo biloba L. leaves, protected mice against convulsions induced by 4-O-methylpyridoxine (MPN). To elucidate the mechanism of the anticonvulsant activity of bilobalide, this study examined the effect of bilobalide on MPN-induced changes in the levels of gamma-aminobutyric acid (GABA) and glutamate, and in the activity of glutamic acid decarboxylase (GAD) in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels and GAD activity in the hippocampus and cerebral cortex were significantly enhanced by bilobalide treatment (30 mg/kg, p.o., for 4 days) alone. MPN significantly decreased GABA levels and GAD activity in the three brain regions tested compared with those in the control. Pretreatment with bilobalide effectively suppressed the MPN-induced reduction in GABA levels and GAD activity in the hippocampus and cerebral cortex. On the other hand, there were no significant differences in the glutamate levels in the three regions despite various treatments. These results suggested that bilobalide prevents MPN-induced reduction in GABA levels through potentiation by bilobalide of GAD activity, and this effect of bilobalide contributes to its anticonvulsant effect against MPN-induced convulsions.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.     

Relationship between bound and free amino acids in the brain of growing rats]

Total pool of glutamate, glutamine and GABA in the hemispheres increases during postnatal life of rats, the increase being due to that in free and bound forms of amino acids. In the cerebellum of 1-day rats, the content of free and bound glu, gln asp, GABA, bound ala and free gly is lower, whereas the level of free glu and ala, bound gly is higher than in mature animals. To the end of the 1st week, total amino acid content decreases, except GABA, which is increased. Aminon acid content begins to increase at the 21th and 28th days of postnatal life.     

EFFECTS OF DI-n-PROPYLACETATE,AN ANTICONVULSIVE COMPOUND,ON GABA METABOLISM1

The effects on GABA metabolism of an anticonvulsant drug, di-n-propylacetate (DPA), were studied. Given intraperitoneally DPA increases the brain GABA content and does not change its biosynthesis from glutamic acid. However, it inhibits in vitro both glutamate decarboxylase and aminobutyrate transaminase (GABA-T) activities. The inhibition is more pronounced on the GABA-T and this observation might explain the increase of GABA level.     

The Synthesis of Neuroactive Amino Acids from Radioactive Glucose and Glutamine in the Rat Retina: Effects of Light Stimulation

The effect of light stimulation in vitro on the labelling of neuroactive amino acids derived from [14C]glucose or [14C]glutamine in the rat retina has been studied. [14C]Glutamine, at 700 microM, provided about 50% of the tissue pools of glutamate, aspartate, and GABA; and the labelling of these decreased on light stimulation, both in the photoreceptor cells (glu and asp) and in the inner retina (glu, asp, and GABA). In contrast, there were no significant changes in the entry of label derived from [14C]glucose, although similar trends were apparent in the data obtained for the photoreceptor cell layer. The pools may, therefore, be separate. Other results support the contention that glucose is the principal energy source for the retina, its entry into non-amino acid derivates being decreased on light stimulation.     

On the role of GABA in vertebrate polyamine metabolism

4-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrate brain, is formed not only by decarboxylation of glutamic acid but also directly from putrescine. Two pathways can be shown to operate in vertebrates: oxidative deamination by diamine oxidase and transformation of putrescine into monoacetylputrescine with subsequent oxidative deamination of this intermediate by monoamine oxidase. Monoacetylation and oxidation degradation of the acetyl derivatives is most probably a common pathway of the polyamines. The formation of spermic acid and putreanine from spermine and spermidine, respectively, seems analogous to the reaction of putrescine with diamine oxidase. Apart from metabolic transformation of the polyamines to GABA, there are indirect interrelations with potential regulatory functions. A variety of agents able to influence brain GABA metabolism induce changes of the activity of the decarboxylases involved in polyamine metabolism and alterations of cerebral putrescine concentrations. These interrelations could be important in the control of local cerebral protein metabolism. The excessive transformation of putrescine to GABA in early neural development suggests a role in cellular differentiation.     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.     

Brain glutamate metabolism during metabolic alkalosis and acidosis.

Glutamate modifies ventilation by altering neural excitability centrally. Metabolic acid-base perturbations may also alter cerebral glutamate metabolism locally and thus affect ventilation. Therefore, the effect of metabolic acid-base perturbations on central nervous system glutamate metabolism was studied in pentobarbital-anesthetized dogs under normal acid-base conditions and during isocapnic metabolic alkalosis and acidosis. Cerebrospinal fluid transfer rates of radiotracer [13N]ammonia and of [13N]glutamine synthesized de novo via the reaction glutamate+NH3-->glutamine in brain glia were measured during normal acid-base conditions and after 90 min of acute isocapnic metabolic alkalosis and acidosis. Cerebrospinal fluid [13N]ammonia and [13N]glutamine transfer rates decreased in metabolic acidosis. Maximal glial glutamine efflux rate jm equals 85.6 +/- 9.5 (SE) mumol.l-1 x min-1 in all animals. No difference in jm was observed in metabolic alkalosis or acidosis. Mean cerebral cortical glutamate concentration was significantly lower in acidosis [7.01 +/- 0.45 (SE) mumol/g brain tissue] and tended to be larger in alkalosis, compared with 7.97 +/- 0.89 mumol/g in normal acid-base conditions. There was a similar change in cerebral cortical gamma-aminobutyric acid concentration. Within the limits of the present method and measurements, the results suggest that acute metabolic acidosis but not alkalosis reduces glial glutamine efflux, corresponding to changes in cerebral cortical glutamate metabolism. These results suggest that glutamatergic mechanisms may contribute to central respiratory control in metabolic acidosis.     

THE GAMMA-AMINOBUTYRIC ACID (GABA) SYSTEM IN BRAIN DURING ACUTE AND CHRONIC ETHANOL INTOXICATION

Abstract— The effects of acute and chronic ethanol intoxication on the GAGA system of rats have been investigated. Under the terminal conditions provoked. by ethanol (6–8 g/kg, i.p.) the brain GABA content sharply increased. There was a simultaneous decrease of 35–40% in the glutamate decarboxylase (GAD) activity of the cerebellum and cerebral hemispheres. In contrast, the transaminase, GABA-T was either unchanged, or it increased: by 28% only in cerebellum and by 1.5–2.0–fold in liver and kidney. It is suggested that effects of acute ethanol intoxication at different doses (2–8 g/kg) on the brain GABA system is connected with the phases of the functional condition of the CNS and a disturbance of homeostatic function. Chronic ethanol consumption caused a decrease in brain GABA. an increase of GAD activity in cerebellum and cerebral hemispheres, and no change in GABA-T activity. The activity of this last enzyme was increased 1.5–2.0-fold in liver and kidneys of rats consuming a diet containing 10% ethanol daily. A 50-fold purified preparation of GABA-T obtained from pig brain was inhibited by butanol-l and propanol-1 (0.03–0.6m) with no effect of ethanol. It is suggested that the mechanisms involved in the ethanol effect on nervous cells are linked with the GABA system and the phases of the functional condition of the CNS.     

Glutamic acid and gamma-aminobutyric acid neurotransmitters in central control of breathing.

We review recent cross-disciplinary experimental and theoretical investigations on metabolism of the amino acid neurotransmitters glutamic acid and gamma-aminobutyric acid (GABA) in the brain during hypoxia and hypercapnia and their possible role in central control of breathing. The roles of classical modifiers of central chemical drive to breathing (H+ and cholinergic mechanisms) are summarized. A brief perspective on the current widespread interest in GABA and glutamate in central control is given. The basic biochemistry of these amino acids and their roles in ammonia and bicarbonate metabolism are discussed. This review further addresses recent work on central respiratory effects of inhibitory GABA and excitatory glutamate. Current understanding of the sites and mechanisms of action of these amino acids on or near the ventral surface of the medulla is reviewed. We focus particularly on tracer kinetic investigations of glutamatergic and GABAergic mechanisms in hypoxia and hypercapnia and their possible role in the ventilatory response to hypoxia. We conclude with some speculative remarks on the critical importance of these investigations and suggest specific directions of research in central mechanisms of respiratory control.     

GABA content and glutamic acid decarboxylase activity in brain of Huntington's chorea patients and control subjects.

—GABA contents are significantly decreased in the caudate nucleus, putamen-globus pallidus, substantia nigra, and occipital cortex in autopsied brain from Huntington's chorea patients, as compared to values in the same regions from control subjects who have died without neurological disease. Homocarnosine levels are lower in choreic than in control brain, but only in the putamen-globus pallidus and the cerebellar cortex are the differences significant. Activity of the enzyme which synthesizes GABA, glutamic acid decarboxylase, is reduced in the brains of some choreic patients, but may be equally low in brain of control subjects, even though the latter exhibit normal brain GABA content. Low glutamic acid decarboxylase activity in autopsied human brain is not uniquely characteristic of Huntington's chorea. No evidence was found in this study for an inhibitor of glutamic acid decarboxylase in choreic brain, nor for the presence of an isoenzyme with decreased affinity for glutamate. GABA aminotransferase, the enzyme which degrades GABA, was equally active in control and choreic brain; therefore, increased activity of this enzyme cannot account for the low brain GABA levels in Huntington's chorea.     

13C Nuclear Magnetic Resonance Evidence for γ-Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.     

Ethanol and other CNS depressants decrease GABA synthesis in mouse cerebral cortex and cerebellum in vivo

GABA synthesis in mouse brain $\text{in vivo}$ was estimated by measuring the rate of GABA accumulation one hour after inhibition of GABA degradation using the selective and irreversible antagonism of GABA-transaminase by gabaculine. Using this method we found that acute and repeated ethanol administration lead to a potent depression of gabaculine induced enhancement of GABA levels in mouse brain cerebellum and cerebral cortex. Alcohol, in the absence of gabaculine had no effect on steady state GABA levels. These results demonstrate potent effects of ethanol on the dynamics of GABA metabolism which are compatible with a GABA like effect of ethanol.     

FORMATION OF GABA FROM PUTRESCINE IN THE BRAIN OF FISH (SALMO IRIDEUS GIBB.)

Abstract— It was demonstrated after intraperitoneal and intracerebral injections of [1,4-¹⁴C]-putrescine.2 HCl that GABA is formed in vivo in the trout brain via a pathway in which glutamic acid is not an intermediate. Intraperitoneal and intracerebral injections of both thiosemicarbazide and 3-mercaptopropionic acid had no measurable effects on GABA concentration, transformation of glutamic acid into GABA in vivo , or on glutamate de-carboxylase activity in the brain within the first 3 h after the application of the inhibitors. Only a small decrease in concentration of pyridoxal phosphate was noticed in the fish brain after thiosemicarbazide administration. The relatively high concentrations of pyridoxal phosphate in the trout brain may be one of the reasons for the ineffectiveness of thiosemicarbazide in inhibiting glutamate decarboxylase in vivo. After intracerebral injections of [1-¹⁴C]GABA, a half-life of 7 h was determined for GABA. The slow turnover rate of GABA in trout brain, which can be assumed from this observation, may give a further explanation of the ineffectiveness of the glutamate decarboxylase inhibitors in lowering the GABA content ot fish brain within a few hours.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.     

THE EFFECTS OF 4-HYDROXYBUTYRIC ACID ON THE BIOSYNTHESIS OF AMINO ACIDS IN THE CENTRAL NERVOUS SYSTEM

—The effect of 4-hydroxybutyrate (GHB) on cerebral glucose metabolism has been studied. GHB increases the glucose level, decreases the lactate concentration and diminishes the incorporation of glucose carbon into glutamic acid, glutamine, aspartic acid and GABA in the brain of the rat in a state of general anaesthesia. The data reported here suggest that GHB interferes in the metabolism of glucose in brain.     

Alterations in Uptake and Release Rates for GABA, Glutamate, and Glutamine During Biochemical Maturation of Highly Purified Cultures of Cerebral Cortical Neurons, a GABAergic Preparation

This study demonstrates that virtually homogenous cultures of mouse cerebral neurons, obtained from 15-day-old embryos, differentiate at least as well as cultures which in addition contain astrocytes. This was indicated by glutamate decarboxylase activity which within 2 weeks rose from a negligible value to twice the level in the adult mouse cerebral cortex, and by a gamma-aminobutyric acid (GABA) uptake rate which quadrupled during the second week in culture and reached higher values than in brain slices. Within the same period, the GABA content increased four to five times to 75 nmol/mg protein, and a potassium-induced increase in [14C]GABA efflux became apparent. Although the development was faster than in vivo, optimum differentiation required maintenance of the cultures beyond the age of 1 week. Uptake and release rates for glutamate and glutamine underwent much less developmental alteration. At no time was there any potassium-induced release of radioactivity after exposure to [14C]glutamate, and the glutamate uptake was only slightly increased during the period of GABAergic development. This indicates that exogenous glutamate is not an important GABA precursor. Similarly, glutamine uptake was unaltered between days 7 and 14, although a small potassium-induced release of radioactivity after loading with glutamine suggests a partial conversion to GABA.