Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

Pentylenetetrazole inhibits glutamate dehydrogenase and aspartate aminotransferase, and stimulates GABA aminotransferase in homogenates from rat cerebral cortex

The mechanism by which pentylenetetrazole provokes convulsions in animals has been investigated by measuring its influence in vitro on the activities of several enzymes of glutamate metabolism in rat brain homogenates. Pentylenetetrazole does not affect the specific activities of glutamine synthetase, glutaminase, or glutamate decarboxylase; it inhibits those of glutamate dehydrogenase and aspartate aminotransferase, and stimulates that of gamma-aminobutyric acid (GABA) aminotransferase. The overall consequence of the action of pentylenetetrazole on the activities of these enzymes should be an increase in the concentration of glutamate and a decrease in that of GABA. This modulation of glutamate and GABA metabolism by pentylenetetrazole could contribute to the triggering of convulsions.     

Distribution of Metabolites and Enzymes of Nitrogen Metabolism between the Mesophyll and Paraveinal Mesophyll of Non-Nodulated Glycine max

The distribution of amino acids and key enzymes involved innitrogen metabolism was determined in mesophyll cells (MC),mesophyll protoplasts (MP), and paraveinal mesophyll protoplasts(PVMP) isolated from fully expanded trifoliolate leaves of non-nodulatedsoybean. Qualitative and quantitative differences were foundin the distribution of amino acids, with MP containing the highestconcentrations. Activity of nitrate reductase, glycolate oxidase,glutamine synthetase and glutamate dehydrogenase was measuredin both tissue types and differences in activities between thetissue types were seen. PVMP had high glutamate dehydrogenaseactivity when compared to MP. Activities of glycolate oxidaseand glutamine synthetase were much higher in MP on a protoplastbasis while nitrate reductase activity was similar between thetwo protoplast types. These results, on the distribution ofmetabolites and associated enzymes, are discussed as to theirpossible significance to nitrogen metabolism in the soybeanleaf. Key words: Amino acids, glutamate dehydrogenase, Glycine max, nitrate reductase, nitrogen metabolism, paraveinal mesophyll, protoplasts     

Aspartate aminotransferase in synaptic and nonsynaptic mitochondria: differential effect of compounds that influence transient hetero-enzyme complex (metabolon) formation

The enzyme aspartate aminotransferase (AAT) has a number of key roles in astrocytes and neurons in brain. An understanding of the regulation of AAT is important since AAT is involved in many aspects of glutamate metabolism including the synthesis of neurotransmitter glutamate. Mitochondrial AAT binds to a protein and lipids on the inner mitochondrial membrane and also forms a number of transient hetero-enzyme complexes with other enzymes. These complexes serve to facilitate metabolism by essentially channeling substrates and cofactors to other enzymes within the complex. The association and dissociation of transiently formed hetero-enzyme complexes may modulate enzyme activity in "real time" since these complexes are dynamically influenced by changes in the concentration of a number of key metabolites. The influence of several effectors that modulate AAT activity, either directly, or by altering the binding of AAT to mitochondrial lipids, or the association/dissociation into transient hetero-enzyme complexes was determined. The addition of palmitate, malate, citrate, glutamate, bovine serum albumin and Mg(2+) modulated AAT activity differently in synaptic and nonsynaptic mitochondria from brain. These findings suggest that AAT activity and also glutamate metabolism, may be regulated in part, by metabolites that influence binding of the enzyme to lipids or proteins in the inner mitochondrial membrane and/or the association/dissociation of transient hetero-enzyme complexes. This may have a role in the compartmentation of glutamate metabolism in brain.     

Histochemical demonstration of glutamate dehydrogenase and phosphate-activated glutaminase activities in semithin sections of the rat retina

Summary The activities of the glutamate metabolizing enzymes phosphate-activated glutaminase (PAG) and glutamate dehydrogenase (Gldh) are demonstrated in semithin sections of the rat retina. Highest activities of both enzymes are found in the photoreceptor inner segments, PAG additionally in the outer plexiform layer and Gldh in the inner plexiform layer and in mueller glial cells. Although their non randomly distribution makes a role in neurotransmitter metabolism possible, their high activities in inner segments point towards the general problem of the functional interpretation of both molecules.     

Histochemical demonstration of glutamate dehydrogenase and phosphate-activated glutaminase activities in semithin sections of the rat retina.

The activities of the glutamate metabolizing enzymes phosphate-activated glutaminase (PAG) and glutamate dehydrogenase (Gldh) are demonstrated in semithin sections of the rat retina. Highest activities of both enzymes are found in the photoreceptor inner segments, PAG additionally in the outer plexiform layer and Gldh in the inner plexiform layer and in mueller glial cells. Although their non randomly distribution makes a role in neurotransmitter metabolism possible, their high activities in inner segments point towards the general problem of the functional interpretation of both molecules.     

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.     

Cerebral Ammonia Metabolism in Hyperammonemic Rats

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.     

Nitrogen-assimilating enzymes in land plants and algae: phylogenic and physiological perspectives

An important biochemical feature of autotrophs, land plants and algae, is their incorporation of inorganic nitrogen, nitrate and ammonium, into the carbon skeleton. Nitrate and ammonium are converted into glutamine and glutamate to produce organic nitrogen compounds, for example proteins and nucleic acids. Ammonium is not only a preferred nitrogen source but also a key metabolite, situated at the junction between carbon metabolism and nitrogen assimilation, because nitrogen compounds can choose an alternative pathway according to the stages of their growth and environmental conditions. The enzymes involved in the reactions are nitrate reductase (EC 1.6.6.1-2), nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13-14, 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.2-4), aspartate aminotransferase (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Many of these enzymes exist in multiple forms in different subcellular compartments within different organs and tissues, and play sometimes overlapping and sometimes distinctive roles. Here, we summarize the biochemical characteristics and the physiological roles of these enzymes. We also analyse the molecular evolution of glutamine synthetase, glutamate synthase and glutamate dehydrogenase, and discuss the evolutionary relationships of these three enzymes.     

Ammonia Assimilation in Rumen Bacteria: A Review

In the rumen bacteria, ammonia as the end product of nitrogen is incorporated into carbon skeleton (α-ketoglutarate) to yield glutamine and glutamate which are important nitrogen donors in nitrogenous compounds metabolism in cells. The enzymes glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase are involved in these processes. Some experimental results have proven that the global nitrogen regulation system may participate in the regulation of assimilation of ammonia in rumen bacteria. This review offers a current perspective on the pathways and key enzymes of ammonia assimilation in rumen bacteria with the possible molecular regulation strategy, while points out the further research direction.