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.     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

Bmcc1s interacts with the phosphate-activated glutaminase in the brain

Bmcc1s, a brain-enriched short isoform of the BCH-domain containing molecule Bmcc1, has recently been shown to interact with the microtubule-associated protein MAP6 and to regulate cell morphology. Here we identified kidney-type glutaminase (KGA), the mitochondrial enzyme responsible for the conversion of glutamine to glutamate in neurons, as a novel partner of Bmcc1s. Co-immunoprecipitation experiments confirmed that Bmcc1s and KGA form a physiological complex in the brain, whereas binding and modeling studies showed that they interact with each other. Overexpression of Bmcc1s in mouse primary cortical neurons impaired proper mitochondrial targeting of KGA leading to its accumulation within the cytoplasm. Thus, Bmcc1s may control the trafficking of KGA to the mitochondria.     

Effect of starvation or streptozotocin-diabetes on phosphate-activated glutaminase of different rat brain regions

Phosphate-activated glutaminase (PAG) was assayed in homogenates of brain cerebellum, hippocampus or striatum from normal, starved for 48 h or 120 h or streptozotocin-diabetic rats. Only the hippocampal enzyme was increased (47%) by diabetes. Starvation had no effect in any of the regions studied. PAG of synaptosomes or of non-synaptosomal mitochondria from the hippocampus was also increased by 48% and 22% respectively in diabetes. PAG of synaptosomes from the cortex, the cerebellum, or the striatum or of the non-synaptosomal mitochondria from the cortex were not affected by diabetes or prolonged (120h) starvation. A suggestion is presented that peripheral insulin, indirectly, may regulate PAG activity in a specific region of the rat brain.Abbreviations used PAG phosphate-activated glutaminase - LDH lactate dehydrogenase - s.a. specific activity     

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.     

Different fixative-resin combinations and the immunocytochemical demonstration of immunoglobulins (kappa light chains) in semithin sections

Summary The effect of six different epoxy resins on the immunostaining of kappa light chains in tonsilar tissue which had been fixed by one of three fixing variants is described. The results show that not only may a specific resin influence the reaction but this may be enhanced by adopting a particular fixative-resin combination.     

Glutamate transport,glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.     

The use of Quetol 651 for the post-embedding immunohistochemical demonstration of gamma-aminobutyric acid on semithin sections

Summary Quetol 651 was used as an embedding medium for the demonstration of aminobutyric acid (GABA) in semithin sections by the peroxidase—anti-peroxidase method. In order to demonstrate the immunoreactivity, the embedding medium was partially dissolved using absolute ethanol containing 0.8–1m NaOH or KOH for 5–7 min. The experimental procedure was elaborated by testing the GABAergic sites in the endings surrounding the small neurones of the anterior exterolateral nucleus of a mormyrid fish and in the pyramidal cells of the electrosensory lateral line lobe of gymnotoid fish by applying anti-GAD (glutamic acid decarboxylase) antiserum. To test the general validity of the use of Quetol 651, GABAergic sites were also identified in the central nervous system of an insect, the honey bee, with anti-GABA and anti-GAD antisera. The intensity of labelling revealed by immunoperoxidase applied to Quetol 651-embedded semithin sections, demonstrated high precision and gave good resolution for light microscopical observations.     

Effect of neonatal monosodium glutamate on the activities of glutamate dehydrogenase and aminotransferases in the circumventricular organs of rat brain

Summary Glutamate (Glu) the major amino acid in mammalian brain and most dietary proteins possesses neurotransmitter as well as neurotoxic properties. We administered monosodium glutamate (MSG) 4 mg/g bwt, sc on postnatal day (PND) 1 through 10 to rats on alternate days or daily and sacrificed them on PND 45 or PND 90 respectively. The activities of glutamate dehydrogenase and aminotransferases were evaluated in the circumventricular organs of brain. Results show that neonatal MSG produces alterations in glutamate metabolism in blood-brain-barrier deficient regions.     

Effects of mitochondrial swelling and calcium on phosphate-activated glutaminase in pig renal mitochondria

The effects of mitochondrial swelling and calcium have been used to study the possible function of the glutamine transporter in regulating glutamine hydrolysis. Salt-induced swelling of pig renal mitochondria and an iso-osmotic mixed salt solution and swelling caused by reducing the osmolarity of the incubation medium, are accompanied by activation of glutamine hydrolysis. Regulation of the glutaminase activity by salt-induced mitochondrial swelling is likely to have physiological importance, similar to the regulation of hepatic glutaminase by changing the matrix volume, that has been described by others. 0.1-1.0 mM calcium stimulates glutamine hydrolysis and the calcium activation curve follows Michaelis-Menten kinetics. The calcium activation is reversible, it is unaffected by phosphate, high glutamine and mitochondrial calcium uptake, as well as by sonication and the activation is calmodulin independent. The calcium activation is additive to that of swelling. Similar to calcium, hypo-osmotic swelling mainly increases the apparent Vmax for glutamine, whereas the apparent Km is little changed, indicating that the effects are primarily on the phosphate-activated glutaminase itself rather than on the glutamine transporter. Furthermore, calcium which activates glutamine hydrolysis, inhibits glutamine uptake into the mitochondria and so does alanine having no effect on glutamine hydrolysis. Therefore, it is indicative that glutamine transport is not rate limiting for glutamine hydrolysis.     

Demonstration of dehydrogenase activities in paraffin sections.

The possibility of demonstrating the activity of respiratory enzymes in paraffin sections was studied. Unfixed pieces of nervous tissue were incubated at 4 degrees C, 20 degrees C, 37 degrees C and 56 degrees C for various periods ranging from 1 to 24 hours. After dehydration, the tissue pieces were mounted in paraffin. The paraffin sections obtained there of were then tested with respect to the range of penetration of the substrate into the incubated tissue samples (as judged from the resulting histoenzymic reaction), and for the distinctness with which the localization of the histochemic reaction could be assessed. From the results it may be concluded that it is possible, under well defined conditions, to demonstrate the activity of dehydrogenases in paraffin sections. The resulting morphological pictures permit a much better localization of the histoenzymic reaction products than those obtained from cryostat sections. Optimal results are obtained when tissue fragments, about 1 mm in diameter are incubated for 24 hours at 4 degrees C.