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1.
Biochim Biophys Acta Biomembr ; 1862(9): 183362, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32445746

ABSTRACT

Here, both neuroprotectants, i.e. cholesterol depletion of the plasma membrane of rat brain nerve terminals (synaptosomes) using methyl-ß-cyclodextrin (MßCD) and deep/propound hypothermia, were analyzed during their combined administration and regarding additive neuroprotective effect. The extracellular synaptosomal level of L-[14C]glutamate significantly increased after treatment with MßCD in both deep and profound hypothermia. Cholesterol depletion gradually enhanced inhibiting effect of deep and profound hypothermia on glutamate uptake and "excitotoxic" transporter-mediated release of L-[14C]glutamate. A decrease in L-[14C]glutamate release via heteroexchange from nerve terminals in deep and profound hypothermia was enhanced by cholesterol deficiency that confirmed previous result. Fluorometric studies with probes NR12S and DCVJ revealed oppositely directed effects of cholesterol depletion and hypothermia on synaptosomal membrane lipid order and microviscosity showing that cholesterol depletion can normalise up to the control hypothermia-induced increase in microviscosity, but not the lipid order of the synaptosomal membrane. Dynamics of changes in exocytosis in nerve terminals, which involved membrane fusion stage, was different from transporter-dependent ones. Hypothermia did not augment effects of cholesterol depletion on exocytotic L-[14C]glutamate release and lowering cholesterol enhanced the impact of deep, but not profound hypothermia on this parameter. Therefore, dual benefit of combined neuroprotection was demonstrated. Cholesterol depletion enhanced neuroprotective effects of hypothermia intensifying inhibition of "excitotoxic" transporter-mediated glutamate release and can normalise a hypothermia-induced increase in microviscosity of the synaptosomal membrane. This feature is prospective in mitigation of side effects of therapeutic hypothermia, and also for brain conservation preserving normal physical and chemical properties of the cellular membranes.


Subject(s)
Cerebral Cortex , Cholesterol/metabolism , Hypothermia , Neuroprotection , Synaptosomes/metabolism , Animals , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Hypothermia/metabolism , Hypothermia/therapy , Male , Rats , Rats, Wistar , beta-Cyclodextrins/pharmacology
2.
Anal Chim Acta ; 1022: 113-123, 2018 Aug 31.
Article in English | MEDLINE | ID: mdl-29729731

ABSTRACT

An excess of the excitatory neurotransmitter, glutamate, in the synaptic cleft during hypoxia/ischemia provokes development of neurotoxicity and originates from the reversal of Na+-dependent glutamate transporters located in the plasma membrane of presynaptic brain nerve terminals. Here, we have optimized an electrochemical glutamate biosensor using glutamate oxidase and developed a biosensor-based methodological approach for analysis of rates of tonic, exocytotic and transporter-mediated glutamate release from isolated rat brain nerve terminals (synaptosomes). Changes in the extracellular glutamate concentrations from 11.5 ±â€¯0.9 to 11.7 ±â€¯0.9 µΜ for 6 min reflected a low tonic release of endogenous glutamate from nerve terminals. Depolarization-induced exocytotic release of endogenous glutamate was equal to 7.5 ±â€¯1.0 µΜ and transporter reversal was 8.0 ±â€¯1.0 µΜ for 6 min. The biosensor data correlated well with the results obtained using radiolabelled L-[14C]glutamate, spectrofluorimetric glutamate dehydrogenase and amino acid analyzer assays. The blood plasma glutamate concentration was also tested, and reliability of the biosensor measurements was confirmed by glutamate dehydrogenase assay. Therefore, the biosensor-based approach for accurate monitoring rates of tonic, exocytotic and transporter-mediated release of glutamate in nerve terminals was developed and its adequacy was confirmed by independent analytical methods. The biosensor measurements provided precise data on changes in the concentrations of endogenous glutamate in nerve terminals in response to stimulation. We consider that the glutamate biosensor-based approach can be applied in clinics for neuromonitoring glutamate-related parameters in brain samples, liquids and blood plasma in stroke, brain trauma, therapeutic hypothermia treatment, etc., and also in laboratory work to record glutamate release and uptake kinetics in nerve terminals.


Subject(s)
Biosensing Techniques/methods , Blood Chemical Analysis/methods , Brain/cytology , Glutamic Acid/blood , Glutamic Acid/metabolism , Synaptosomes/metabolism , Animals , Electrochemistry , Exocytosis , Glutamate Dehydrogenase/metabolism , Rats , Rats, Wistar
3.
Cell Mol Neurobiol ; 36(8): 1229-1240, 2016 Nov.
Article in English | MEDLINE | ID: mdl-26886753

ABSTRACT

Extracellular/intracellular L-[14C]glutamate exchange and conservativeness of the extracellular level of L-[14C]glutamate was analyzed in isolated rat brain nerve terminals. L-Glutamate-, DL-threo-ß-hydroxyaspartate (DL-THA)-, and D-aspartate-induced increase in the ambient level of L-[14C]glutamate or D-[3H]aspartate was evaluated comparatively. 100 µM "cold" nonradiolabeled L-glutamate, DL-THA, D-aspartate extruded a quarter of radioactivity from L-[14C]glutamate-preloaded synaptosomes for 6 min. The similar results were obtained with L-glutamate-evoked extracellular/intracellular redistribution of D-[3H]aspartate. Contribution of presynaptic glutamate receptors to an increase in the extracellular L-[14C]glutamate level was evaluated using receptor agonists NMDA, AMPA, and kainate (100 µM), and it consisted of less than 5 % of total accumulated label. The existence of the efficient extracellular/intracellular glutamate exchange, and so dynamic glutamate gradient across the plasma membrane of nerve terminals was demonstrated. A two-substrate kinetic algorithm that included transporter reversal was considered. The extracellular level of L-[14C]glutamate and D-[3H]aspartate in nerve terminals depended on the amount of exogenous substrates of glutamate transporter available. Taking into account that L-glutamate, DL-THA, and D-aspartate are the substrates of glutamate transporters, and also the similarity in their effectiveness in the establishment of new extracellular level of the neurotransmitters, the central role of glutamate transporters in permanent glutamate turnover in nerve terminals was demonstrated.


Subject(s)
Glutamic Acid/metabolism , Presynaptic Terminals/metabolism , Synaptic Membranes/metabolism , Animals , Aspartic Acid/metabolism , Glutamate Dehydrogenase/metabolism , Male , Rats , Rats, Wistar , Synaptosomes/metabolism
4.
Talanta ; 135: 67-74, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25640127

ABSTRACT

Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na(+)-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor.


Subject(s)
Biosensing Techniques , Glutamic Acid/analysis , Synaptosomes/metabolism , Animals , Brain/cytology , Electrodes , Glutamate Dehydrogenase/metabolism , Glutamic Acid/metabolism , Male , NAD/metabolism , Oxidoreductases , Platinum , Rats , Rats, Wistar
5.
Mol Cell Neurosci ; 58: 95-104, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24321453

ABSTRACT

Ferritin, an iron storage protein, is present in the serum and cerebrospinal fluid, has receptors on the cell surface, able to penetrate the brain-blood barrier, can be secreted from the cells, and leaks from destroyed cell in insult and brain trauma. The effect of exogenous ferritin on the key characteristic of glutamatergic neurotransmission was assessed in rat brain nerve terminals (synaptosomes). Exogenous ferritin (80 µg/ml, iron content 0.7%) significantly increased the ambient level of L-[(14)C]glutamate (0.200±0.015 versus 0.368±0.016 nmol/mg of protein) and endogenous glutamate (fluorimetric glutamate dehydrogenase assay) in the nerve terminals. This increase was not a result of augmentation of tonic release because the velocity of tonic release of L-[(14)C]glutamate was not changed significantly in ferritin-treated synaptosomes as compared to the control. Ferritin caused a decrease in synaptic vesicle acidification that was shown using fluorescent dye acridine orange. Iron-dependence of the effects of ferritin was analyzed with apoferritin (0.0025% residual iron). Apoferritin weakly affected the proton electrochemical gradient of synaptic vesicles but increased the ambient level and decreased the initial velocity of uptake of L-[(14)C]glutamate by synaptosomes, nevertheless these effects were ~30% lesser than those caused by ferritin. Exogenous ferritin can provoke the development of excitotoxicity increasing the ambient level of glutamate and lowering synaptic vesicle acidification and glutamate uptake in the nerve terminals, however these effects are not completely iron-dependent. Thus, in the CNS exogenous ferritin can act as a modulator of glutamate homeostasis in iron-dependent and iron-independent manner.


Subject(s)
Apoferritins/pharmacology , Brain/metabolism , Glutamic Acid/metabolism , Membrane Potentials , Presynaptic Terminals/metabolism , Protons , Synaptic Vesicles/metabolism , Animals , Brain/drug effects , Brain/physiology , Hydrogen-Ion Concentration , Male , Presynaptic Terminals/drug effects , Presynaptic Terminals/physiology , Rats , Rats, Wistar , Synaptic Transmission , Synaptic Vesicles/drug effects , Synaptosomes/drug effects , Synaptosomes/metabolism , Synaptosomes/physiology
6.
Biochim Biophys Acta ; 1822(10): 1553-61, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22713486

ABSTRACT

BACKGROUND: In our earlier work, a reduction of cholesterol content increased the extracellular glutamate level in rat brain nerve terminals (synaptosomes) that was a result of the lack of transporter-mediated glutamate uptake. The aim of this study was to assess transporter-mediated release of glutamate from cholesterol-deficient synaptosomes. In stroke, cerebral hypoxia/ischemia, and traumatic brain injury, the development of neurotoxicity is provoked by enhanced extracellular glutamate, which is released from nerve cells mainly by glutamate transporter reversal - a distinctive feature of these pathological states. METHODS: Laser scanning confocal microscopy, spectrofluorimetry, radiolabeled assay, and glutamate dehydrogenase assay. RESULTS: Cholesterol acceptor methyl-ß-cyclodextrin (15mM) reduced the cholesterol content in the synaptosomes by one quarter. Transporter-mediated glutamate release from synaptosomes: 1) stimulated by depolarization of the plasma membrane; 2) by means of heteroexchange with competitive transportable inhibitor of glutamate transporters dl-threo-ß-hydroxyaspartate; 3) in low [Na(+)] medium; and 4) during dissipation of the proton gradient of synaptic vesicles by the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP); was decreased under conditions of cholesterol deficiency by ~24, 28, 40, and 17%, respectively. CONCLUSIONS: A decrease in the level of membrane cholesterol attenuated transporter-mediated glutamate release from nerve terminals. Therefore, lowering cholesterol may be used in neuroprotection in stroke, ischemia, and traumatic brain injury which are associated with an increase in glutamate uptake reversal. This data may explain the neuroprotective effects of statins in these pathological states and provide one of the mechanisms of their neuroprotective action. However, beside these disorders, lowering cholesterol may cause harmful consequences by decreasing glutamate uptake in nerve terminals.


Subject(s)
Amino Acid Transport System X-AG/metabolism , Brain/drug effects , Cell Membrane/drug effects , Cholesterol/metabolism , Glutamic Acid/metabolism , Nerve Endings/drug effects , Neuroprotective Agents/pharmacology , Animals , Brain/metabolism , Brain Injuries/drug therapy , Brain Injuries/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Membrane/metabolism , Hypoxia-Ischemia, Brain/drug therapy , Hypoxia-Ischemia, Brain/metabolism , Male , Membrane Potentials/drug effects , Nerve Endings/metabolism , Protons , Rats , Rats, Wistar , Sodium/metabolism , Stroke/drug therapy , Stroke/metabolism , Synaptic Vesicles/drug effects , Synaptic Vesicles/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , beta-Cyclodextrins/pharmacology
7.
Neurochem Int ; 55(8): 724-31, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19631248

ABSTRACT

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.


Subject(s)
Brain/metabolism , Glutamic Acid/metabolism , Hypergravity/adverse effects , Presynaptic Terminals/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Acridine Orange/pharmacokinetics , Adenosine Triphosphate/metabolism , Animals , Brain/pathology , Brain/physiopathology , Calcium Signaling/physiology , Cell Compartmentation/physiology , Cognition Disorders/etiology , Cognition Disorders/metabolism , Cognition Disorders/physiopathology , Cytoplasm/metabolism , Disease Models, Animal , Exocytosis/physiology , Fluorescent Dyes/pharmacokinetics , Magnesium/metabolism , Male , Membrane Fusion/physiology , Presynaptic Terminals/pathology , Rats , Rats, Wistar , Synaptic Vesicles/pathology , Synaptosomes
8.
Adv Space Res ; 33(8): 1362-7, 2004.
Article in English | MEDLINE | ID: mdl-15803628

ABSTRACT

The biochemical basis underlying the effects of altered gravity on the process of nervous signal transmission is not clear. We have investigated the effect of hypergravity stress (created by centrifugation of rats at l0 g for 1 h) on the basal and stimulated release of L-[14C]glutamate (a chemical transmitter of excitatory signals) from isolated rat brain nerve terminals (synaptosomes). It has been shown that the hypergravity stress exerted a different influence on the Ca(2+)-dependent and the Ca(2+)-independent component of neurotransmitter release. The Ca(2+)-dependent L-[14C]glutamate release evoked by potassium chloride was equal to 14.4 +/- 0.7% of total synaptosomal label for control animals and 6.2 +/- 1.9% for animals, exposed to hypergravity (P < or = 0.05) and was more than twice decreased as a result of the hypergravity stress. We observed no statistically significant difference in the Ca(2+)-independent component of L-[14C]glutamate release. For control group and animals exposed to the hypergravity stress it was equal to 7.7 +/- 2.8% and 12.9 +/- 2.0%, respectively. We have also investigated the effect of the hypergravity stress on the activity of high-affinity Na(+)-dependent glutamate transporters. Km and Vmax of L-[14C]glutamate uptake have been determined. The maximal velocity of glutamate uptake was decreased as a result of hypergravity loading, but no difference in the Km values between control rats and hypergravity exposed animals was observed. These findings indicate that hypergravity stress alters neurotransmitter reuptake and exocytotic neurotransmitter release processes.


Subject(s)
Brain/metabolism , Glutamic Acid/metabolism , Glutamic Acid/pharmacokinetics , Hypergravity , Synaptosomes/metabolism , Animals , Calcium/metabolism , Centrifugation , Male , Potassium Chloride , Rats , Rats, Wistar , Synaptic Transmission
9.
J Gravit Physiol ; 9(1): P25-6, 2002 Jul.
Article in English | MEDLINE | ID: mdl-14703670

ABSTRACT

We have investigated the effects of altered gravity on the kinetic parameters of glutamate transport activity. We observed no differences in Km values for cerebellum and cerebral hemisphere nerve terminals (synaptosomes) between control rats- 18,2 +/- 7,6 micromoles (cerebellum), 10,7 +/- 2,5 micromoles (cerebral hemispheres) and animals exposed to hypergravity- 23,3 +/- 6,9 micromoles (cerebellum), 6,7 +/- 1,5 micromoles (cerebral hemispheres). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate in cerebellum and cerebral hemispheres was not sensitive to hypergravity stress. The maximal velocity of L-[14C]-glutamate uptake (Vmax) reduced for cerebellum synaptosomes from 9,6 +/- 3,9 nmol/min/mg of protein in control group to 7,4 +/- 2,0 nmol/min/mg of protein in animals, exposed to hypergravity stress. For cerebral hemisphere synaptosomes the maximal velocity significantly decreased from 12,5 +/- 3,2 nmol/min/mg of protein to 5,6 +/- 0,9 nmol/min/mg of protein, respectively.

10.
Neurophysiology ; 34(2-3): 118-9, 2002.
Article in English | MEDLINE | ID: mdl-14983839

ABSTRACT

The effects of hypergravity stress on L-[14C]-glutamate release from synaptosomes obtained from the rat brain and on the kinetic parameters of high-affinity glutamate transport activity were investigated. We found that hypergravity stress affected only the Ca(2+)-dependent component of L-[14C]-glutamate release. It did not modify the transporter affinity, but the maximum rate of uptake dropped from 12.5 +/- 3.2 to 5.6 +/- 0.9 nmol/min/mg of protein (in control rats and in animals subjected to hypergravity, respectively).


Subject(s)
Brain/metabolism , Calcium/physiology , Glutamic Acid/pharmacokinetics , Hypergravity , Synaptosomes/metabolism , Animals , Centrifugation , Culture Media , Potassium Chloride/pharmacokinetics , Rats
11.
Patol Fiziol Eksp Ter ; (4): 53-5, 1994.
Article in Russian | MEDLINE | ID: mdl-7700705

ABSTRACT

Malonate was tested for its effects on rat hepatic metabolism of [1,4-14C]-succinic acid and [1-14C]-glycerol. The administration of malonic acid did not limit [1,4-14C]-succinate metabolism and change the rate of radioactive labelling from [1-14C]-glycerol to the total lipid fraction, as well as the fraction of phospholipids and triacylglycerides. However, there was a reduction in the use of glycerol as a precursor of higher fatty acids and cholesterol. It is suggested that the detected effect of malonate is due to its action on oxidative decarboxylation of pyruvic acid.


Subject(s)
Liver/drug effects , Malonates/pharmacology , Animals , Female , Liver/metabolism , Rats , Rats, Wistar
12.
Eksp Klin Farmakol ; 55(6): 56-8, 1992.
Article in Russian | MEDLINE | ID: mdl-1305881

ABSTRACT

Malonic acid showed a protective action on rats in acute hypoxic hypoxia. The antihypoxic effect of this compound is unassociated with its action on succinate dehydrogenase and may be mediated through the changes in hormonal metabolic regulation.


Subject(s)
Hypoxia/prevention & control , Malonates/therapeutic use , Acute Disease , Animals , Atmosphere Exposure Chambers , Blood/drug effects , Blood/metabolism , Brain/drug effects , Brain/metabolism , Drug Evaluation, Preclinical , Female , Hypoxia/metabolism , Liver/drug effects , Liver/metabolism , Rats , Rats, Wistar
13.
Ukr Biokhim Zh (1978) ; 64(6): 76-9, 113, 1992.
Article in Russian | MEDLINE | ID: mdl-1488816

ABSTRACT

Malonate regulates the liver metabolism and promotes stimulation of the fatty acids synthesis and pyruvate oxidation inhibition. The latter is particularly significant in regulation of aerobic processes and responsible for antihypoxic action of malonate.


Subject(s)
Lipids/biosynthesis , Liver/drug effects , Malonates/pharmacology , Animals , Carbon Radioisotopes , Female , In Vitro Techniques , Liver/metabolism , Rats , Rats, Wistar , Time Factors , Tritium
14.
Ukr Biokhim Zh (1978) ; 63(5): 57-62, 1991.
Article in Russian | MEDLINE | ID: mdl-1788875

ABSTRACT

Cathepsin D preparations have been isolated from the heart of healthy animals and stress-surviving rats by the method of affine chromatography with the hemoglobin-biogel-P300 sorbent. To analysis of the obtained data permits concluding that acute stress stimulates activation of the catalytic function of cathepsin D in the heart. But the period after the stress accompanied by the consecutive proteolysis rate reduction, that can be explained, probably, by a change in enzyme conformation. The concentration of Ca2+ (10(-6), 10(-5) M) and cAMP (10(-7), 10(-6) M) exert a regulating influence on the cathepsin D activity in the heart in acute stress period and after it.


Subject(s)
Cathepsin D/metabolism , Myocardium/enzymology , Pain/enzymology , Stress, Psychological/enzymology , Animals , Kinetics , Protein Conformation , Rats , Rats, Inbred Strains , Reference Values , Time Factors
16.
Ukr Biokhim Zh (1978) ; 57(3): 69-71, 1985.
Article in Russian | MEDLINE | ID: mdl-4040675

ABSTRACT

It is established that leucine aminopeptidase activity during emotional-algesic stress increases in the brain hemispheres, left ventricle and liver of the rats as compared to that of intact animals. Maximum activation of the enzyme in the brain and liver is detected for the first two days after the stress while in the heart - for the whole period of the total stress damages volume development. A conclusion is drawn that the shifts observed in leucine aminopeptidase activity during emotional-algesic stress affect the methionine-enkephalin and leucin-enkephalin ratio.


Subject(s)
Brain/enzymology , Leucyl Aminopeptidase/metabolism , Stress, Psychological/enzymology , Animals , Humans , Liver/enzymology , Myocardium/enzymology , Rats , Rats, Inbred Strains , Time Factors
17.
Ukr Biokhim Zh (1978) ; 57(2): 15-8, 1985.
Article in Russian | MEDLINE | ID: mdl-4039861

ABSTRACT

Emotional-algesic stress causes essential changes in the protein metabolism of cerebral hemispheres. These changes may be of great importance for the functioning of the brain and cause the disturbances of the higher nervous activity when the organism is influenced by the emotional stress factors.


Subject(s)
Brain/metabolism , Nerve Tissue Proteins/metabolism , Stress, Psychological/metabolism , Animals , Humans , Rats
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