Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes

The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h. The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+. The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration. It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.     

Effect of castration on the high affinity glutamate transporter in rat hypothalamic and cortical synaptosomes

High affinity transport of glutamic acid has been studied in cortical and hypothalamic synaptosomes from castrated male rats and compared to normal controls. For hypothalamic synaptosomes, both initial velocity of uptake and Va (apparent maximal velocity) were found to be about one-third lower in the castrated animals. Kt (glutamate concentration giving Va/2), however, was reduced by only 5%.Initial velocity of uptake in cortical synaptosomes was measured as a function of both sodium and glutamate concentration. Reductions in uptake subsequent to castration were found to be much less for cortical synaptosomes (2–15%) than for hypothalamic synaptosomes. Fit for these data to various models for the sodium dependence of transport resulted in the same minimal best fit model as that found for control animals. Thus castration does not alter the fundamental nature of the mechanism by which carrier, sodium, and glutamate interact in the process of transport. However quantitative changes were found to occur, as reflected in the best fit constants. These constants were used along with the rate equation for the minimal best fit model to calculate certain parameters which were then used to delineate the quantitative changes in the transporter following castration. A neuroregulatory role for glutamate in gonadotropin secretion has been recently proposed; the present study now provides additional information on the relationship between reproductive function and one aspect of glutamatergic synaptic function, namely, the high affinity transport system.     

Age-related changes in the glutamate metabolism of cerebral cortical slices from rats

The glutamate metabolism in cerebral cortical slices from young (12 weeks) and aged (100 weeks) rats was studied. A highly significant reduction of low affinity glutamate uptake and its metabolism in the aged rats by 14.5% of the amount of the young rats was observed (Pº0.001). When the ratio of the radioactivity of respective metabolite divided by the sum total radioactivity of overall glutamate metabolites was compared, the incorporation to aspartate was significantly small (P<0.01), while that of the CO₂ liberated and the GABA synthesized were increased (P<0.05). These results support the hypothesis that in cerebral cortical slices from aged rats the transamination of glutamate is suppressed and its decarboxylation is enhanced despite markedly reduced low affinity glutamate uptake into the cerebral cortex. The difference could be explained by the fact that cerebral cortical slices from aged rats are more vulnerable to anoxia than those from young rats when exposed during slice preparation.     

Influence of methionine sulfoximine on the photosynthesis of isolated chloroplasts

Methionine sulfoximine provided at a concentration which inhibits photosynthesis in intact leaves (10 mM) had no significant influence on the rate of photosynthesis of isolated pea leaf chloroplasts. In contrast, ammonium, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and D,L-glyceraldehyde all strongly inhibited the photosynthesis of isolated chloroplasts. We conclude that low concentrations of methionine sulfoximine (up to 10 mM) have no direct effect on the photosynthetic process.     

Effects of anticonvulsant drugs on calcium transport and polyphosphoinositide metabolism in rat cortical synaptosomes

1. Anticonvulsants: phenytoin, phenobarbital, carbamazepine and valium at concentrations of 10-100 microM had a significantly inhibitory effect on both K+-stimulated Ca2+-uptake and 32Pi incorporation into phospholipids of rat cortical synaptosomes. 2. Other anticonvulsant, valproic acid, at concentration upto 100 microM had no effect on these two events. 3. Our results suggest that there is a link between Ca2+-influx and polyphosphoinositide turnover in synaptosomes, and this link may relate to the inhibitory effect of these drugs on neurotransmitter release mechanisms of this preparation.     

Cholesterol as a key player in the balance of evoked and spontaneous glutamate release in rat brain cortical synaptosomes

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals.     

The toxicity induced by phosphinothricin and methionine sulfoximine in rice leaves is mediated through ethylene but not abscisic acid

The possible role of ethylene and abscisic acid (ABA) in regulating thetoxicity of detached rice leaves induced by phosphinothricin (PPT) andmethionine sulfoximine (MSO), both known to be glutamine synthetase (GS)inhibitors, was studied. During 12 h of incubation, PPT and MSOinhibited GS activity, accumulated NH₄ ⁺ and inducedtoxicity of detached rice leaves in the light but not in darkness. PPT and MSOtreatments also resulted in an increase of ethylene production and ABA contentin a light dependent way. Addition of fluridone, an inhibitor of ABAbiosynthesis, reduced ABA content in rice leave but did not preventNH₄ ⁺ toxicity of rice leaves induced by PPT and MSO.Cobalt ion, an inhibitor of ethylene biosynthesis, affected PPT- andMSO-inducedtoxicity of detached rice leaves but had no effect on PPT- and MSO-inducedNH₄ ⁺ accumulation. Results suggest that ethylene but notABA may be responsible for PPT- and MSO-induced toxicity of detached riceleaves.     

A unified assay for six enzymes of glutamate metabolism.

A single assay system has been developed for six enzymes of glutamate metabolism: glutamate dehydrogenase, glutaminase, asparate aminotransferase, γ-aminobutyrate aminotransferase, alanine aminotransferase, and glutamate decarboxylase. The first five are assayed by coupling them to Escherichia coli glutamate decarboxylase and measuring the release of ¹⁴CO₂ from radioactive substrates. Glutamate decarboxylase is assayed directly. The assays are simple, use but one technique, and require very little working time. At a reasonable cost per assay, they are considerably more sensitive than other commonly used assays for the same enzymes. The sensitivity of the assay at a fixed price increases as the substrate concentration decreases.     

Ammonia accumulation and inhibition of photosynthesis in methionine sulfoximine treated spinach

Ammonia accumulation and photosynthetic rate inhibition took place when spinach leaf tissue was supplied with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. This effect was observed in the absence of significant inorganic nitrogen reduction or an exogenous source of ammonia. Both the time lag prior to the initial photosynthetic rate decrease and the rate of that decrease depend on the O₂ and MSO concentrations supplied to the leaf tissue. However, the total rate of ammonia accumulation was similar at both 20% and 2.2% O₂. The decline in photosynthetic rate was not caused by stomatal closure but may be a result of ammonia toxicity. The data point out the importance of glutamine synthetase in preventing the poisoning of leaf metabolism by ammonia generated internally through processes not involved in net nitrogen assimilation. The rapidity of the action of MSO in suppressing photosynthesis was unexpected and should not be overlooked in interpreting data from other experiments involving that inhibitor. MSO shows promise as a tool for investigating C-N flow, particularly during photorespiration.