Kynurenate production by cultured human astrocytes.

In the rodent brain, astrocytes are known to be the primary source of kynurenate (KYNA), an endogenous antagonist of both the glycine(B) and the alpha7 nicotinic acetylcholine receptor. In the present study, primary human astrocytes were used to examine the characteristics and regulation of de novo KYNA synthesis in vitro. To this end, cells were exposed to KYNA's bioprecursor L-kynurenine, and newly formed KYNA was recovered from the extracellular milieu. The production of KYNA was stereospecific and rose with increasing L-kynurenine concentrations, reaching a plateau in the high microM range. In an analogous experiment, astrocytes also readily produced and liberated the potent, specific glycine(B) receptor antagonist 7-chlorokynurenate from L-4-chlorokynurenine. KYNA synthesis was dose-dependently reduced by L-leucine or L-phenylalanine, two amino acids that compete with L-kynurenine for cellular uptake, and by aminooxyacetate, a non-specific aminotransferase inhibitor. In contrast, KYNA formation was stimulated by 5 mM pyruvate or oxaloacetate, which act as co-substrates of the transamination reaction. Aglycemic or depolarizing (50 mM KCl or 100 microM veratridine) conditions had no effect on KYNA synthesis. Subsequent studies using tissue homogenate showed that both known cerebral kynurenine aminotransferases (KAT I and KAT II) are present in astrocytes, but that KAT II appears to be singularly responsible for KYNA formation under physiological conditions. Taken together with previous results, these data suggest that very similar mechanisms control KYNA synthesis in the rodent and in the human brain. These regulatory events are likely to influence the neuromodulatory effects of astrocyte-derived KYNA in the normal and diseased human brain.

In vivo glutamine hydrolysis in the formation of extracellular glutamate in the injured rat brain.

Hydrolysis of extracellular glutamine as a potential source of increased extracellular glutamate in the quinolinic acid (QUIN)-injured brain of the unanesthetized, free-moving rat was examined by microdialysis and HPLC analysis. Injury was initiated by injection of 100 nmoles of QUIN into the hippocampus. Immediately postinjury or 24 hr postinjury, the injection site was perfused with artificial cerebrospinal fluid + (14)C-glutamine to measure its conversion to (14)C-glutamate. L-trans-pyrrolidine-2,4-dicarboxylate (L-PDC), a glutamate uptake inhibitor, was added to the perfusate to enhance the detection of extracellular (14)C-glutamate. QUIN injury was followed by an immediate increase in extracellular glutamate that persisted 24 hr later. When (14)C-glutamine was added to the perfusate, a significant amount of (14)C-glutamate was recovered, and it was greater following QUIN injury than in control animals (P < 0.001). Up to 32% of the extracellular (14)C-glutamine was converted to (14)C-glutamate following QUIN injury. Considering the high concentration of glutamine normally present in the extracellular fluid, glutamine hydrolysis is a potential and important source for the increase in extracellular glutamate after neuronal injury in vivo.

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis.

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

Effect of alpha-ketoisocaproate and leucine on the in vivo oxidation of glutamate and glutamine in the rat brain.

Leucine and alpha-ketoisocaproate (alpha-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or alpha-KIC on the oxidation of L-[U-14C]glutamate and L-[U-14C]glutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of [14C]glutamate and [14C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and alpha-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate on glutamine. Infusion of 0.5 mM alpha-KIC increased [14C]glutamate oxidation approximately 2-fold; higher concentrations of alpha-KIC did not further stimulate [14C]glutamate oxidation. The enhanced oxidation of [14C]glutamate may be attributed to the function of alpha-KIC as a nitrogen acceptor from [14C]glutamate yielding [14C]alpha-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [14C]glutamine oxidation was not stimulated as much as [14C]glutamate oxidation and only increased at 10 mM alpha-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [14C]glutamate or [14C]glutamine. In maple syrup urine disease elevated alpha-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.

Induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic AMP and dexamethasone.

Arginine is an intermediate in the elimination of excess nitrogen and is the substrate for nitric oxide synthesis. Arginine synthesis has been reported in brain tissue. We have studied the activity of the arginine biosynthetic enzymes argininosuccinate synthetase and argininosuccinate lyase in dexamethasone and/or dibutyryl cyclic AMP treated rat astrocyte cultures. Argininosuccinate lyase activity was stimulated by treatment with either effector and an additive effect was obtained when both agents were added simultaneously. Argininosuccinate synthetase was also increased in dexamethasone treated astrocytes. The effect of dibutyryl cyclic AMP on argininosuccinate synthetase was variable, suggesting a role for additional factors in its regulation as compared to argininosuccinate lyase. Regulation of arginine synthesis in astrocytes may be important to insure that arginine is not limiting for nitric oxide synthesis in neural tissue.

Use of intracellular versus extracellular specific activities in calculation of glutamine metabolism in astrocytes: effect of dibutyryl cyclic AMP.

The rate of glutaminase-dependent metabolism of glutamine in intact astrocytes was determined under conditions in which the extracellular concentration of glutamine was varied between 0.2 and 3.2 mM glutamine for control and dibutyryl cyclic AMP (dBcAMP)-treated cells. Glutamine metabolism by intact cells increased with increasing extracellular glutamine when calculations were based on the extracellular specific activity of glutamine. However, when the rate was based on the intracellular specific activity of glutamine, the rate of glutamine metabolism was independent of the media glutamine concentration. Similar results were obtained when cells were treated with dBcAMP, although the rates were approximately twice as high compared to untreated cells. The rate of formation of 14CO2 from [1-14C]glutamine and [1-14C]glutamate, based on the extracellular specific activities, were 93 +/- 5 and 40 +/- 4 nmol/mg protein/h, respectively. Oxidation rates based on the experimentally determined intracellular specific activity of glutamine and glutamate were 144 +/- 8 and 209 +/- 18 nmol/mg protein/h, respectively. In dBcAMP-treated astrocytes, the oxidation rates were higher than in untreated cells. These studies demonstrate that determination of the specific activity of compounds inside the cell aids in the interpretation of metabolic studies with intact cells and that both the initial steps of glutamine metabolism and the rate of 14CO2 formation from 14C-glutamine via the TCA cycle were increased in dBcAMP-treated astrocytes.

Elevation of amino acids in the interstitial space of the rat brain following infusion of large neutral amino and keto acids by microdialysis: alpha-ketoisocaproate infusion.

alpha-Ketoisocaproate was infused into the brain of free-moving, awake rats by microdialysis to create a microenvironment similar to that found in maple syrup urine disease. The eluate of the probe was analyzed for amino acids to determine if alpha-ketoisocaproate was transaminated to leucine and if the amino acid homeostasis was altered. The interstitial levels of leucine were increased up to 11-fold and other large neutral amino acids were increased 2- to 3-fold indicating an active branched chain keto acid transaminase activity and enhanced hetero-exchange across cell membranes. The elevation of large neutral amino acids in the interstitial space is discussed in terms of the synthesis of leucine and neurotransmitters in maple syrup urine disease.

Elevation of amino acids in the interstitial space of the rat brain following infusion of large neutral amino and keto acids by microdialysis: leucine infusion.

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Monitoring in vivo oxidation of 14C-labelled substrates to 14CO2 by brain microdialysis.

Cultured brain cells oxidize glucose and numerous alternate substrates to CO2 for energy production, however, the importance of these observations to the intact brain have not been established. We have adapted in vivo brain microdialysis procedures to measure the rate of 14CO2 formation from 14C-glutamate, 14C-glutamine, and 14C-glucose in the hippocampus of awake unanesthetized free-moving rats. Two, 9 and 16 days after surgery (to implant guide cannulae) microdialysis probes were inserted into the guide cannulae and perfused with artificial CSF containing either 14C-glutamate, 14C-glutamine or 14C-glucose. Dialysate fractions were collected during 20 min intervals for determination of 14CO2. The amount of labelled 14CO2 increased for 40 to 60 min and then plateaued and remained relatively constant for at least 6 hours. When the probe was removed from the hippocampus and inserted into a vial containing non-radioactive CSF, 14CO2 production dropped rapidly. The quantity of 14CO2 recovered from glutamate was greater than from glucose or glutamine reflecting pool sizes, uptake characteristics and point of entry into oxidative pathways. The microdialysis system was verified by using model systems with cultured astrocytes suspended in media to simulate the brain. The present results indicate brain microdialysis may be used to study the role of alternate substrates in specific brain regions under varying physiological states.

Functional intracellular glutaminase activity in intact astrocytes.

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the "functional" intracellular phosphate-activated glutaminase activity was measured by the formation of 3H2O from L-[2-3H]glutamine (Anal. Biochem. 127:134-142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of 14CO2 release from L-[1-14C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1 mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of 14CO2 release from L-[1-14C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 microM glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic exposure to subcutaneously implanted methylxanthines. Differential elevation of A1-adenosine receptors in mouse cerebellar and cerebral cortical membranes.

Upregulation of brain adenosine receptors in DBA/2J mice as affected by theophylline and caffeine, adenosine antagonists, was examined following subcutaneous drug implantation to ensure chronic exposure. Scatchard analysis of binding to membranes of cerebral cortex and cerebellum from individual mice showed a differential upregulation of (-)-N6-R-[G-3H]phenylisopropyladenosine ([3H]-L-PIA) binding density by theophylline. After 14 days of exposure to theophylline (serum concentration of 1.2 +/- 0.01 micrograms/ml measured by HPLC analysis), the Bmax for L-PIA binding to cerebellar membranes increased 22% over the control mice (statistically significant at P less than 0.01 level). Theophylline had no effect on the Bmax for L-PIA binding to cerebral cortical membranes. The observed increases in Bmax values of cerebellar (13.2%) and cerebral cortical membrane binding (14.2%) on chronic exposure to caffeine (7.1 +/- 0.5 micrograms/ml) were not statistically significant at the P less than or equal to 0.05 level. Neither methylxanthine affected the dissociation constant, KD, for L-PIA. The increased potential for adenosine receptor upregulation by theophylline compared to caffeine following chronic, low level exposure suggests that caffeine treatment for sleep apnea may be preferred to the standard theophylline therapy.

Lack of a sustained effect on catecholamines or indoles in mouse brain after long term subcutaneous administration of caffeine and theophylline.

Short term administration of methylxanthines has been reported to alter levels and turnover rates of brain catecholamines and indoles. In the present study continuous administration of caffeine and theophylline was achieved by subcutaneous implantation of silastic tubing filled with powdered methylxanthines. Serum levels of caffeine and theophylline were monitored daily for 2 weeks by high performance liquid chromatography (HPLC) and averaged 35 microM and 7 microM, respectively. After 2 weeks of continuous exposure to methylxanthines the dopamine level and turnover rate were unaltered from control in the neostriatum, hypothalamus and cortex. Likewise the level and turnover of norepinephrine were unaltered from control in the cerebellum, hypothalamus and cortex. Also unaffected were the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA), serotonin and 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus and cortex. These results indicate that in mice the continuous exposure to methylxanthines has no long lasting effect on monoamine neurotransmitters in the brain.

Glutamine: a major energy source for cultured mammalian cells.

Cultured mammalian cells have two primary mechanisms for obtaining energy necessary for growth: carbohydrate metabolism to lactate and glutamine oxidation to CO2. In tissue culture medium containing both glucose and glutamine, the contribution of glutamine oxidation to the energy requirement ranges between 30 and 50%. As the glucose concentration is decreased, or when glucose is replaced by other carbohydrates, the rate of glutamine oxidation increases and glutamine becomes the sole energy source for cultured cells. The rate of glutamine oxidation is regulated by the presence of glucose. The apparent absolute requirement for glucose or other carbohydrates in tissue culture medium is related to its role in anabolic reactions rather than in energy production. Oxidation of glucose, fatty acids, or ketone bodies does not contribute significantly to the energy needs of cultured mammalian cells. The data also suggest that consideration should be given to glutamine as an important energy source in vivo.

Comparison of the oxidation of glutamine, glucose, ketone bodies and fatty acids by human diploid fibroblasts.

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts ws studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, D-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.