SUBCELLULAR LOCALIZATION OF N-ACETYL-ASPARTYL-GLUTAMATE, N-ACETYL-GLUTAMATE AND GLUTATHIONE IN BRAIN

Abstract— The subcellular distribution of N-acetyl-aspartate, N-acetyl-aspartyl-glutamate, N-acetyl-glutamate and glutathione (reduced) was investigated. Lactate dehydrogenase, potassium, glutamate and aspartate were employed as markers of the cytoplasmatic compartments. Fumarate hydratase and choline acetyltransferase were used as mitochondrial and synaptosomal markers respectively.
Our data show that the highest concentrations of NAA, NAGA, NAAGA and glutathione were localized in the supernatant with a smaller peak in the crude mitochondrial (P₂) fraction. On subfractionating P₂, NAA was distributed similarly to aspartate and K⁺ with a peak in the synaptosome (B) fraction, while glutathione and NAAGA were localized in the mitochondrial fraction. NAA, aspartate and K⁺ were more readily released than glutathione and NAAGA from their particulate form on exposure to hypo-osmotic conditions.     

THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO-NIGRAL PATHWAY

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices. Unilateral transection of the striato-nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1-¹⁴C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U-¹⁴C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U-¹⁴C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side. Incubation of slices from substantia nigra with β-mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Neurotoxicological Examination of the Piglet Brain after Prenatal and Postnatal Exposure to Trichlorfon

Piglets were given 10 repeated doses of the orga-nophosphorus compound trichlorfon during the postnatal period in order to examine the effect on the brain development (Experiment 1). Following prenatal exposure to trichlorfon, the ability of a presumptive hypoplastic cerebellum and cerebrum at birth, to regenerate postnatally was investigated (Experiment 2). Administration of repeated doses of trichlorfon postnatally was accompanied by only small changes in brain weights, morphology and transmitter enzyme activity (choline acetyltransferase, glutamate decarboxylase, aromatic amino acid decarboxylase) in 35 days old piglets. Animals exposed prenatally, and sacrificed at the age of 35 days, showed a significant increase in brain weights and enzyme activities. The animals did, however, not reach control values in cerebral weight, cerebellar weight or total enzyme activities. Morphological changes still showed regional loss of Purkinje cells in the cerebellum. The study clearly indicated that the pig brain was less vulnerable to trichlorfon in the postnatal period of development than when exposed to the compound prenatally.     

The Acute Effect of Ammonium Acetate on Levels of Amino Acids in the Intact and Decorticated Rat Neostriatum

Unilateral frontal cortex ablations were performed in rats so that the glutamate terminals in the ipsilateral rostral neostriatum were removed. At 1 or 7 days later, intraperitoneal injections of ammonium acetate induced different changes in amino acid concentrations in the intact and deafferentated neostriatum. After 1 day, the level of glutamate decreased only in the intact side, whereas that of glutamine increased and that of aspartate decreased to the same extent on both sides following ammonia injection. After 7 days, the glutamate level decreased more in the intact than the decorticated side in both nonconvulsing and convulsing rats. The concentration of alanine increased most in the intact neostriatum, whereas glutamine levels increased and aspartate levels decreased to the same extent on both sides in nonconvulsing and convulsing rats. The results indicate that ammonia has a more pronounced effect on neuronal than glial glutamate pools.     

Choline acetyltransferase binding to and release from membranes

1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

The ontogeny of the uptake systems for glutamate,GABA, and glycine in synaptic vesicles isolated from rat brain

The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.Special issue dedicated to Dr. Morris H. Aprison.     

Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide

A fragment of the amyloid beta protein, &#103 A(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that &#103 A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, &#103 A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by &#103 A(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to &#103 A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 &#148 &#109 ) - and hydroxyl ( &#148 OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of &#148 OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of &#148 OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to &#103 A(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that &#103 A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 &#148 &#109 can lead to HOCl and further formation of &#148 OH, which both have a cytotoxic potential.