Janet Stern

Glutathione turnover in cultured astrocytes: Studies with [15N]glutamate

Abstract: The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20–40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady‐state incubation medium with 0.05 mM L‐cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage

Abstract: We demonstrate by reverse transcriptase‐polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG‐4) expresses the non‐N‐methyl‐d‐aspartate (non‐NMDA) glutamate receptor (GluR) genes GluR2–7, KA‐1, and KA‐2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1–3, GluR5–7, KA‐1, and KA‐2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24‐h exposure to 500 µM kainate or 5 mMl‐glutamate, but not by a 24‐h exposure to up to 10 mMα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA). Damage is prevented by the non‐NMDA GluR channel inhibitor 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA‐preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA‐preferring and possibly also kainate‐preferring GluR channels that are capable of mediating Ca2+‐dependent excitotoxicity and that AMPA‐induced cytotoxicity is blocked by desensitization of AMPA‐preferring GluRs.

Astrocyte leucine metabolism: significance of branched-chain amino acid transamination.

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 ± 3.2 nmol/mg of protein/min and a Km of 449.2 ± 94.9 µM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α‐aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α‐ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 µM with a Vmax of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α‐ketoglutarate ? KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the “buffering” of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu‐cine furnishes α‐NH2 groups for glutamate synthesis via branched‐chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography‐mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2‐15N]glutamine/[15N]leucine suggested that at least one‐fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [16N]‐isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched‐chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α‐ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ∼50% in only 5 min. Extracellular concentrations of α‐ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched‐chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ∼17 times greater than the rate of [1‐14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a‐ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.

The synthesis of messenger RNA without protein synthesis: I. Studies with thymineless strains of Escherichia coli*

Escherichia coli , deficient in the ability to synthesize thymine, uracil and an amino acid, synthesize a small fraction of their normal RNA from uracil in the absence of thymine and the amino acid. The synthesis of even this fraction of RNA is inhibited by concomitant DNA synthesis. Most (75%) of the RNA made appears on the ribosomes and can be degraded on these structures in the presence of inorganic phosphate. When synthesized in the presence of an inducer of β -galactosidase, the RNA appears to permit the rapid synthesis of a small amount of this enzyme in the apparent absence of the inducer. These observations are consistent with the hypothesis that the RNA made in the absence of an essential amino acid and of protein synthesis is largely messenger RNA.

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α‐ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5‐15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2‐15N]glutamine, representing the reductive amination of 2‐oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]‐glutamate to [2‐15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5‐15N]glutamine and stimulated the formation of [2‐15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5‐15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2‐15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2‐15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U‐14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α‐ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the Km of glutamine synthetase for glutamate, which we determined to be ∼7 mM.

The lethality of streptomycin and the stimulation of RNA synthesis in the absence of protein synthesis.

The action of streptomycin and related drugs has been studied with thymineless, arginineless and uracilless strains of Escherichia coli. Many experiments were performed in the absence of arginine, a condition shown to reduce 35SO42−incorporation (mainly protein synthesis) to 0·5% and less of the rate of incorporation during growth. In the absence of arginine and in the presence of thymine, under conditions in which the rate of RNA synthesis is 10 to 15% of that during normal growth, streptomycin initially inhibits and then markedly stimulates RNA synthesis. In a wide variety of conditions, the inception of stimulation coincides closely with the inception of lethality. Also the rate of stimulated synthesis of RNA correlates well with the rate of loss of capacity to form colonies. Such a stimulation of RNA synthesis and correlation with lethality also occurs in the presence of neomycin and kanamycin. The stimulation does not occur in a derived streptomycin-resistant strain in which streptomycin does inhibit slightly the basal level of RNA synthesis occurring in the absence of protein synthesis. Anaerobiosis, which protects against the lethal action of streptomycin, also prevents the stimulation of RNA synthesis, although the internal concentrations of phosphates and of streptomycin during anaerobiosis were not significantly changed as compared to the concentrations of these substances during aerobiosis. Reduction of the phosphate concentration in a medium containing streptomycin reduced the lag before both the beginning of killing and the stimulation of RNA synthesis, and increased the degree of the latter function. Indeed under these conditions, the stimulation produced by streptomycin approached the stimulated rate of synthesis of RNA produced on addition of chloramphenicol. The RNA made under the influence of streptomycin was partially associated with the ribosomal fraction and had a rapid but incomplete turnover; however, the partial degradation of this RNA in the absence of the antibiotic did not permit the restoration of apparently killed bacteria to viability. The incorporation of fluorouracil in the presence or absence of uracil was not lethal in the absence of streptomycin. In the presence of the antibiotic, lethality was increased by the analog, concomitant with the stimulated incorporation of the analog into the new type of RNA. At the beginning of stimulation of RNA synthesis, the capacity to incorporate arginine is reduced about 50%. The formation of the RNA stimulated by the antibiotic in the absence of protein synthesis (measured by uptake of 35SO42−) is coincident with a rapid reduction of the capacity to incorporate arginine to less than 5% of the normal rate. Thus physiological lethality, i.e. the irreversible loss of the ability to make proteins in the absence of streptomycin, occurs without protein synthesis.

Incorporation of Tritiated Galactose into Galactocerebroside by Cultured Rat Oligodendrocytes: Effects of Cyclic Adenosine 3′,5′-Monophosphate Analogues

Abstract: Cells dissociated from the forebrains of 21‐day‐old rats were enriched in oligodendroglia by Percoll gradient centrifugation, seeded on polylysine‐coated surfaces, and cultured in a serum‐containing medium. Incorporation by the cultures of tritium from d‐[3H]galactose into the galactosyl residue of galactocerebroside (galC) increased in an almost linear fashion for 48 h with 1–8 μCi of d‐[3H]galactose (30 mCi/μmol) per milliliter medium. Treatment for 2 days (day 1–3 after seeding) with 10−4M or 10−3M dibutyryl cyclic adenosine 3′,5′‐monophosphate (db cyclic AMP) or 10−4M 8‐bromo cyclic AMP stimulated galC radiolabelling. Incorporation of d‐[3H]galactose into galC during a terminal 48‐h radiolabelling period was not stimulated when the cells were continuously treated with these cyclic AMP analogues for 8 rather than 2 days.

Inositol uptake by cultured isolated rat Schwann cells

The uptake of radiolabeled myo-inositol by Schwann cells isolated from the sciatic nerve of 2-4 day old rats was found to occur by a saturable, sodium-dependent phlorizin-inhibited mechanism with an estimated Km of 30 microM. The system was inhibited by galactose and glucose but not by galactitol. At high concentrations of myo-inositol, a diffusion-like process appeared to be functional. The characteristics of the saturable system are very similar to those of myo-inositol uptake by the endoneural fascicle preparation of sciatic nerve.

Effects of palmitate on astrocyte amino acid contents

The effects of palmitate on intracellular and extracellular amino acid concentrations of cultured astrocytes was studied. Exposure of astrocytes to either 0.72 mM or 0.36 mM palmitate was associated with a significant reduction in the intracellular pool of glutamine and taurine. In contrast, the intracellular concentration of histidine, glycine, citrulline, isoleucine and leucine were increased in the presence of 0.72 mM palmitate. Comparable changes in the extracellular amino acid pool were not observed. The data suggest that palmitic acid, which accumulates in the brain during periods of anoxia, alters the metabolism of several amino acids in cultured astrocytes. These changes may be of significance in terms of the pathophysiology of a stress such as anoxia.