Effects Of Methionine Sulfoximine Research Articles

Jaroslava Folbergrová, Dr.Sc. (1932 – 2024)

Jaroslava Folbergrová, an outstanding scientist, passed away on Sunday, January 28 at the age of 91. She was born in 1932 in Nový Bydžov, Czechoslovakia. She studied biochemistry in Charles University, School of Mathematics and Physics in Prague. She started her scientific carrier with studies of the metabolism of ammonia in the brain. She came to the Institute of Physiology, Czechoslovak Academy of Sciences, Prague, in 1958 and she worked there for incredible 65 years till the very end of her life. All the time she was interested in brain metabolism mostly during and after epileptic seizures – at first in Department of Cytochemistry of Nerve Cell, headed by Zdeněk Lodin, where she defended Ph.D. thesis on the „Effect of methionine sulfoximine on brain metabolism“. She defended her „Doctor of Science (Dr.Sc.)“ thesis in 1990 and continued working in this laboratory till 2000 when she entered Department of Developmental Epileptology of the Institute of Physiology of the Czech Academy of Sciences. Her research was further focused on brain metabolism in relation to epileptic seizures and also to brain hypoxia. Two international collaborations were important for her scientific life: Eighteen months spent in 1965-1967 in Washington University in St. Louis, MO where she worked under the leadership of prof. Oliver Lowry, and longlasting collaboration with prof. Siesjo in Lund University, Sweden. Her stage in prof. Lowry’s Institute resulted in four highly cited papers (including one with 255 citations). Repeated visits in Lund led to nearly 50 primary publications in prestigious international journals (some of these papers also reached more than 200 citations). She progressively extended her field of interest to developing brain and the role of oxidative stress in epileptic seizures and their metabolic and functional consequences. Jarka Folbergrová was fully devoted to her scientific work. It was common to see her leaving the institute in the late afternoon with her notebook and a lot of papers to continue in her work at home. She will be missed in the Institute because she very well represented the tradition both at our country and abroad. She was also very friendly person and an inspiring colleague.

Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. The first EEG signal became apparent at ~10min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r> .92) and moderately correlated (r~ -.59) with eGln, but not with eGlu. Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Inhibition of Glutamate Release, but Not of Glutamine Recycling to Glutamate, Is Involved in Delaying the Onset of Initial Lithium-Pilocarpine-Induced Seizures in Young Rats by a Non-Convulsive MSO Dose.

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

Pretreatment with a glutamine synthetase inhibitor MSO delays the onset of initial seizures induced by pilocarpine in juvenile rats

The contribution of glutamatergic transmission to generation of initial convulsive seizures (CS) is debated. We tested whether pretreatment with a glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), affects the onset and progression of initial CS by cholinergic stimulus in juvenile rats. Male rats (24 days old, Sprague Dawley) sequentially received i.p. injections of lithium-carbonate, MSO, methyl-scopolamine, and pilocarpine (Pilo). Pilo was given 150 min after MSO. Animals were continuously monitored using the Racine scale, EEG/EMG and intrahippocampal glutamate (Glu) biosensors. GS activity as measured in hippocampal homogenates, was not altered by MSO at 150 min, showed initial, varied inhibition at 165 (15 min post-Pilo), and dropped down to 11% of control at 60 min post-Pilo, whereas GS protein expression remained unaltered throughout. Pilo did neither modulate the effect of MSO on GS activity nor affect GS activity itself, at any time point. MSO reduced from 32% to 4% the number of animals showing CS during the first 12 min post-Pilo, delayed by ~6 min the appearance of electrographic seizures, and tended to decrease EMG power during ~15 min post-Pilo. The results indicate that MSO impairs an aspect of glutamatergic transmission involved in the transition from the first cholinergic stimulus to the onset of seizures. A continuous rise of extracellular Glu lasting 60 min was insignificantly affected by MSO, leaving the nature of the Glu pool(s) involved in altered glutamatergic transmission undefined.

Involvement of astroglial glutamate–glutamine shuttle in modulation of the jaw‐opening reflex following infraorbital nerve injury

To evaluate the mechanisms underlying orofacial motor dysfunction associated with trigeminal nerve injury, we studied the astroglial cell activation following chronic constriction injury (CCI) of the infraorbital nerve (ION) immunohistochemically, nocifensive behavior in ION-CCI rats, and the effect of the glutamine synthase (GS) blocker methionine sulfoximine (MSO) on the jaw-opening reflex (JOR), and also studied whether glutamate-glutamine shuttle mechanism is involved in orofacial motor dysfunction. GFAP-immunoreactive (IR) cells were observed in the trigeminal motor nucleus (motV) 3 and 14days after ION-CCI, and the nocifensive behavior and JOR amplitude were also strongly enhanced at these times. The number of GS- and GFAP-IR cells was also significantly higher in ION-CCI rats on day 7. The amplitude and duration of the JOR were strongly suppressed after MSO microinjection (m.i.) into the motV compared with that before MSO administration in ION-CCI rats. After MSO administration, the JOR amplitude was strongly suppressed, and the duration of the JOR was shortened. Forty minutes after m.i. of glutamine, the JOR amplitude was gradually returned to the control level and the strongest attenuation of the suppressive effect of MSO was observed at 180min after glutamine m.i. In addition, glutamine also attenuated the MSO effect on the JOR duration, and the JOR duration was extended and returned to the control level thereafter. The present findings suggest that astroglial glutamate-glutamine shuttle in the motV is involved in the modulation of excitability of the trigeminal motoneurons affecting the enhancement of various jaw reflexes associated with trigeminal nerve injury.

Effect of Glutamine Synthetase Inhibition on Brain and Interorgan Ammonia Metabolism in Bile Duct Ligated Rats

Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1 G93A gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan–Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% ( p < 0.01). These results show that in the SOD1 G93A model of neurodegenerative diseases, the concentration of brain glutamate (determined with 1H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

Development of Transfection and High-Producer Screening Protocols for the CHOK1SV Cell System

To date, the FDA has approved 18 monoclonal antibody (MAb) therapeutic drugs with targets ranging from asthma and rheumatoid arthritis to leukemia. Many of these approved products are produced in Chinese hamster ovary cells (CHO) making CHO a significant and relevant host system. We studied the applicability of CHOK1SV cells as a potential host cell line for MAb production in terms of timelines, achievable titers, transfectant stability, and reproducibility. CHOK1SV, developed by Lonza Biologics, is a suspension, protein-free-adapted CHOK1-derivative utilizing the glutamine synthetase (GS) gene expression system. CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants were obtained under the dual selection of methionine sulfoximine (MSX) and glutamine-free media. We examined outgrowth efficiencies, specific productivities, and achievable batch titers of three different IgG MAbs transfected into CHOK1SV. Reducing the MSX concentration in the initial selection medium resulted in a decreased incubation time required for transfectant colonies to appear. Specific productivities of "high-producers" ranged between 11 and 49 pg/c/d with batch titers ranging from 105 to 519 mg/L. Transfectant stability and the effects of MSX also were investigated, which indicated that the addition of MSX was necessary to maintain stable MAb production. Cell growth was stable regardless of MSX concentration.

Effects of Methionine Sulfoximine on the Glutamine and Glutamate Content and Cell Volume in Rat Cerebral Cortical Slices: Involvement of Mechanisms not Related to Inhibition of Glutamine Synthesis

Treatment of cerebral cortical slices with 5 mM ammonium acetate (ammonia) elevated the glutamine (Gln) content and increased cell volume in the slices, in agreement with the postulated contribution of glutamine to hyperammonemic brain edema [Neurochem. Int. 43 (2003) 299]. In this study we show that, unexpectedly, treatment with a glutamine synthetase inhibitor, methionine sulfoximine (MSO) (0.1–5.0 mM) in the absence of ammonia increases Gln content in the slices in a dose-independent manner, to levels higher than those recorded after ammonia treatment. MSO (>0.1 mM) inhibited [ 14C ]Gln uptake in crude cerebral cortical cell membranes (P2 fraction). Since Gln uptake in this preparation was largely facilitated by the Gln efflux-promoting systems ASC and N and less so by the uptake promoting system A, MSO-induced accumulation of Gln could result from inhibition of Gln efflux. MSO did not affect cell volume in the slices, showing that Gln retention is not as a rule a causative factor in cerebral edema. MSO at 5 mM concentration increased cell swelling induced by ammonia, which is consistent with earlier observations pointing to the direct excitotoxic action of MSO in vivo and in vitro. The results emphasize the limits of applicability of MSO as an inhibitor of Gln synthesis in an in vitro system.

Ornithine decarboxylase activity in cerebral post-ischemic reperfusion damage: effect of methionine sulfoximine.

Excessive activation of glutamate receptors via the N-methyl-D-aspartate (NMDA) subtype appears to play a role in the sequence of cellular events which lead to irreversible ischemic damage to neurons. Furthermore, NMDA receptor activation induces a stimulation of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine (PA) biosynthesis. In order to better understand the role of PA we have measured ODC activity and the effect of methionine sulfoximine (MSO), a molecule able to stimulate ODC, on a model of transient cerebral ischemia. There was a significant increase in ODC activity in the rat cerebral cortex during post-ischemic reperfusion. The treatment with MSO induced a significant decrease in cerebral glutamine synthetase activity accompanied by a marked increase in ODC activity. In MSO-pretreated rats there was a significant decrease in the survival rate when compared to untreated ischemic rats.

Complex Roles of Glutamate in the Gibbs—Ng Model of One-trial Aversive Learning in the New-born Chick

NG, K. T., B. S. O'DOWD, N. S. RICKARD, S. R. ROBINSON, M. E. GIBBS, C. RAINEY, W.-Q. ZHAO, G. L. SEDMAN AND L. HERTZ. Complex roles of glutamate in the Gibbs-Ng Model of one-trial aversive learning in the newborn chick. NEUROSCI BIOBEHAV REV 21 [1]45–54, 1997.—Glutamate is the most widespread excitatory transmitter in the CNS and is probably involved in LTP, a neural phenomenon which may be associated with learning and memory formation. Intracerebral injection of large amounts of glutamate between 5 min before and 2.5 min after passive avoidance learning in young chicks inhibits short-term memory, which occurs between 0 and 10 min post-learning in a three-stage model of memory formation first established by Gibbs and Ng [25][Physiol. Behav. 23:369–375; 1979]. This effect may be attributed to non-specific excitation. Blockade of glutamate uptake by l-aspartic acid β-hydroxamate also abolishes this stage of memory, provided the drug is administered within 2.5 min of learning. Interference with either the production of precursors for transmitter glutamate in astrocytes or with glutamate receptors is also detrimental to memory formation, but the effects appear much later. After its release from glutamatergic neurons, glutamate is, to a large extent, accumulated into astrocytes where it is converted to glutamine, which can be returned to glutamatergic neurons and reutilized for synthesis of transmitter glutamate, and partly oxidized as a metabolic substrate. The latter process leads to a net loss of transmitter glutamate which can be compensated for by de novo synthesis of a glutamate precursor α-ketoglutarate (αKG) in astrocytes, a process which is inhibited by the astrocyte-specific toxin fluoroacetate (R. A. Swanson, personal communication). Intracerebral injection of this toxin abolishes memory during an intermediate stage of memory processing occurring between 20 and 30 min post-training [50][Cog. Brain Res. 2:93–102; 1994]. Injection of methionine sulfoximine (MSO), a specific inhibitor of glutamine synthetase, which interferes with the re-supply of transmitter glutamate to neurons by inhibition of glutamine synthesis in astrocytes, has a similar effect. This effect of MSO is prevented by intracerebral injection of glutamate, glutamine, or a combination and αKG and alanine. MSO must be administered before learning, but does not interfere with acquisition since short-term memory remains intact. Administration of either the NMDA antagonist AP5, the AMPA antagonist DNQX, or the metabotropic receptor antagonist MCPF, also induces amnesia. Memory loss in each case does not occur until after 70 min post-training, during a protein synthesis-dependent long-term memory stage which begins at 60 min following learning. However, to be effective, AP5 must be administered within 60 s following learning, MCPG before 15 min post-learning, and DNQX between 15 and 25 min after learning. Together, these findings suggest that learning results in an immediate release of glutamate, followed by a secondary release of this transmitter at later stages of processing of the memory trace, and that one or both of these increases in extracellular glutamate concentration are essential for the consolidation of long-term memory. Since both fluoroacetate and MSO act exclusively on glial cells, the findings also show that neuronal-glial interactions are necessary during the establishment of memory. Copyright © 1996 Elsevier Science Ltd.

Effect of Methionine Sulfoximine on Nitrogen Metabolism and Externally Supplied Ammonium Assimilation in Kidney Bean

L-Methionine sulfoximine (MSO) at concentration 1.25 mM in vivocauses the inhibition of glutamine synthetase (GS) in both roots and leaves of young seedlings of kidney bean following the accumulation of high levels of ammonia and decrease in amounts of free amino acids that is more pronounced in leaves. The inhibition of GS by MSO in leaves in the case of externally supplied 5 mM(15NH4)2SO4assimilation leads to ammonia accumulation and the decrease in the amounts of glutamine and glutamic acid and the intensity of the incorporation of15N into them. In roots the inhibition of GS is not followed by the decrease of15N content into glutamate. It is concluded that the pathway of ammonia primary assimilation in leaves is via GS and glutamate synthase (GOGAT), while in roots glutamate dehydrogenase also plays an important role in this process.

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

Glutamine-synthetase (GS; EC 6.3.1.2) activity and protein levels were measured in crude extracts from Monoraphidium braunii Naegeli, strain 202-7d, cultures grown under different nitrogen sources. Only ammonium and l-glutamine promoted a partial enzyme inactivation, which, in the case of l-glutamine, was accompanied by a significant repression of GS. Methionine sulfoximine (MSX), a strong inhibitor of GS, produced a drastic inactivation of GS which was concomitant with a marked increase in GS protein as measured by rocket immunoelectrophoresis. Such an increase was prevented in the presence of cycloheximide. The effect of the l-glutamine analog on GS activity and protein was partially inhibited if l-glutamine was also added to cell cultures, possibly indicating competition in the transport of these two substances. In addition, the effects of MSX were reversed after longer times when cultures were treated with smaller concentrations of inhibitor. Treatment of cell cultures with azaserine, a specific inhibitor of glutamate synthase, the second enzyme acting in the ammonium assimilation pathway, promoted a strong GS inactivation and a partial repression of this enzyme, which paralleled a specific increase in the intracellular pools of glutamine High-performance liquid chromatography measurements of intracellular amino-acid concentrations showed that glutamine levels correlated negatively with GS concentration. A role for glutamine as a negative effector of GS synthesis is proposed.

Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum.

To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.

Variations in the effects of l-methionine- dl-sulfoximine on the activity of cerebral γ-glutamyl transpeptidase in rats as a function of age

Changes in the activity of γ-glutamyl transpeptidase (GGTP) and the effects of a subacute dose (150 mg/kg b.wt.) of methionine sulfoximine (MSO) on its activity were studied in cerebral cortex, cerebellum and brainstem of rats of 4 different age groups. GGTP activity increased with increasing age between days 10 and 180 after birth and decreased between 180 and 360 days of age in these 3 regions of the brain. Older animals showed convulsions and succumbed to the toxic effects of MSO. In the younger animals, wobbly gait and splayed leggedness were noticed in the earlier time periods and these animals recovered from the toxic effects of the drug. Following the administration of MSO, GGTP activity was suppressed in all the regions (excepting the cerebral cortex and cerebellum of 90-day-old animals). In 10- and 90-day-old animals, there was a complete recovery of enzyme activity which exceeded the control value as the time progressed. No such recovery was observed in 180- and 360-days-old animals. Parallel changes were observed in the GGTP activity and the behavioural pattern of the animals. These results were discussed in relation to the localization of this enzyme in astrocytes and capillaries.

Effect of methionine sulfoximine on pyruvate dehydrogenase, citric acid cycle enzymes and aminotransferases in the subcellular fractions isolated from rat cerebral cortex

The effect of acute and subacute doses of l-methionine- dl-sulfoximine (MSI) were studied on the activities of pyruvate dehydrogenase, enzymes of citric acid cycle and aspartate and alanine aminotransferases in the mitochondria, synaptosomes and cytosol of rat brain. In general, the activities of pyruvate dehydrogenase and of the citric acid cycle enzymes, except malate dehydrogenase (malate → oxaloacetate), were elevated in all 3 subcellular fractions. Malate dehydrogenase activity (malate → oxaloacetate) was suppressed in the mitochondria while the activity of this enzyme in the reverse direction was enhanced in the cytosol. Activities of aspartate and alanine aminotransferases were suppressed under these conditions. As the effects of MSI on these enzymes were similar to those observed upon the administration of ammonium salts, it is suggested that the hyperammonemic state induced by MSI might derange the operation of the malate-aspartate shuttle. Increased activities of citric acid cycle enzymes in the cytosol suggested the existence of a small population of mitochondria which was highly vulnerable either to ammonia or to MSI.

Effect of Methionine Sulfoximine on Asparaginase Activity and Ammonium Levels in Pea Leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.

Effect of methionine-sulfoximine on nitrate uptake and photosynthesis in the cyanobacteriumAnabaena doliolum Bharadwaja

l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited14CO2 fixation and O2 evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH4+) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O2 than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen.

Glycogen synthesis and immunocytochemical study of fructose-1,6-biphosphatase in methionine sulfoximine epileptogenic rodent brain.

The effects of the convulsant methionine sulfoximine (MSO) on the glucose pathway have been investigated in mouse and rat brain. The key gluconeogenic enzyme fructose-1,6-biphosphatase (FBPase) (EC 3.1.3.11) was immunostained by rat anti-FBPase antibody. The rat cortex slices were very lightly stained, almost unstained in controls. After MSO injection, there was a marked staining only in astrocytes (perikarya, processes, and end feet). The activity of this enzyme also increased. MSO induced an increase of 63% in the stability at heating (47 degrees C) and of 36% in the stability at proteolysis (trypsin, 10 micrograms/ml) of FBPase. The convulsant had no effect on the concentrations of the metabolites related to the FBPase-phosphofructokinase step, i.e., fructose-1,6-biphosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate, before, during, or after the convulsions. These results show that the cellular site of glucose pathway impairment induced by MSO in rodent brain is presumably the astroglial cells and that one mechanism of glycogenesis could be the reinforcement of the molecules of FBPase, which enhances gluconeogenesis. A hypothetical diagram of glucose metabolism under the effect of MSO has been proposed.

Regulation of neurotransmitter aspartate metabolism by glial glutamine synthetase.

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.