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Information × Registration Number 0214U002200, 0109U002777 , R & D reports Title Mechanisms of regulation and modulation of the release and active transmembrane transport of neurotransmitters in presynapse in norm and under conditions of development of neuropathology . popup.stage_title Head Borisova Tatiana Alexandrovna, Registration Date 24-01-2014 Organization A.V.Palladin Institute of Biochemistry of National Academy of Sciences popup.description2 The experimental data obtained in the Department of Neurochemistry of Palladin Institute of Biochemistry NAS of Ukraine indicates a close relationship between the level of mitochondrial membrane potential and generation of proton gradient in the membrane of synaptic vesicles, between the efficiency of accumulation of neurotransmitters in the synaptic vesicles and the active transport of neurotransmitters through the plasma membrane of nerve terminals. It has been shown that intracellular processes underlying the stimulation of reverse functioning of membrane transporters in nerve terminals resulted in a considerable depletion of both pools of neurotransmitters, cytosolic and vesicular, followed by blocking of synaptic transmission process. During the reporting period, work was carried out using models of gravitational stress in rats, perinatal hypoxia, deficiency of cholesterol in the membrane structures, cell-free system for studying the process of exocytosis. Methods of radioisotope analysis, fluorescence spectroscopy, laser correlation spectroscopy, confocal microscopy, flow cytometry, the method of registration of ionic conductivity of planar lipid membrane were used. It was found that under gravitational loading of animals there were significant changes in the release of glutamate from presynaptic nerve terminals (synaptosomes), i.e., reduced Ca2+-dependent glutamate release from synaptosomes due to reduction of neurotransmitter content in the synaptic vesicles, whereas increased cytosolic glutamate level due to redistribution of glutamate between vesicular and cytosolic pools. The overall rate of fusion of synaptic vesicles with the plasma membrane in cell-free model system did not change after hypergravity. However, we registered decline in the fusion activity of membrane components and growth of the fusogenic capacity of synaptosomal cytosolic proteins under these conditions. We proved that cholesterol is a potent endogenous modulator of active transport of glutamate in brain nerve terminals and proposed a new mechanism of neuroprotection, which is based on inhibition of transporter-mediated glutamate release by reduction of cholesterol content in the membrane of brain nerve terminals. It was shown that the modulating action of cholesterol acceptor - methyl- -cyclodextrin on glutamate transport was performed by different mechanisms, which depended on methodological protocol of acceptor application. Dissipation of the proton gradient of synaptic vesicles in cholesterol-deficient nerve terminals in the presence of methyl- -cyclodextrin in the incubation medium is an additional factor that inhibits the activity of glutamate transporters of the plasma membrane and increases tonic, Ca2+-independent release of glutamate and its extracellular level and totally inhibits Ca2+-dependent neurotransmitter release. It was suggested that the formation of reactive oxygen species (ROS) under activation of presynaptic glutamate AMPA/NMDA and kainate receptors was coupled with the stimulation of the secretion of GABA from nerve terminals. We showed the different ways of ROS generation under conditions of activation of presynaptic ionotropic glutamate receptors: activation of AMPA/kainate type receptors primarily stimulated mitochondrial ROS generation path, whereas in NMDA-mediated ROS production NADPH-oxidase played a key role. It was revealed that hypoxia and seizures in the perinatal period caused changes in the energy metabolism of brain, increasing the level of ATP by an average of 10 % in all studied structures - cortex, hippocampus and thalamus. Increased extracellular level of GABA was the sustainable long-term effects of perinatal hypoxia. Normalization of extracellular level of GABA was possible at higher levels of ATP, that occurred under conditions when pyruvate was use as the sole energy substrate for cortical and hippocampal neurons. Changes in extracellular GABA level correlated with stimulation of GABA transport into nerve terminal. A significant enhancement in the autoinhibition of GABA secretion in hippocampus and thalamus under conditions of activation of presynaptic GABAB receptors in rats exposed to perinatal hypoxia, suggested changes during postnatal development of the brain under these conditions. A cell-free studies of antiepileptic drug levetiracetam, a specific ligand of synaptic vesicle transmembrane protein SV2, indicated the involvement of SV2 in both homo-/ heterotypic fusion and clustering of synaptic vesicles. Using levetiracetam, we showed that SV2A functioned as a regulator of synaptic vesicle clustering and calcium-regulated fusion of vesicles with the plasma membrane of synaptosomes. Levetiracetam activated calcium-regulated release of GABA from nerve terminals of the cortex, hippocampus and thalamus. Effectiveness of levetiracetam wassignificantly higher in animals exposed to perinatal hypoxia. Applying the model of multivesicular exocytosis, we found that aggregation and docking of synaptic vesicles remained unchanged after partial reduction of cholesterol content in their membranes. However, changes in the kinetics of fusion of synaptic vesicles with each other correlated with a decrease in the level of membrane cholesterol. Oligomeric form of ?A peptide 1-42 inhibited the fusion process of synaptosomal membrane structures at the last stage of regulated exocytosis in vitro. It is known that blood platelets are able to accomplish active accumulation of glutamate and contain high-affinity Na+- dependent glutamate transporters and glutamate receptors. The ability of platelets to accumulate glutamate allows us to believe that they may also participate in the maintenance of extracellular glutamate homeostasis in brain. During the reporting period it was shown that in contrast to brain nerve terminals transporter-dependent and unstimulated glutamate release are not inherent to platelets. The release of glutamate from platelets proceeds by exocytosis only, during platelet activation. It was shown for the first time that amphotericin B and recombinant subunit B of diphtheria toxoid exhibited the formation of long open ionic channels after applying to one side of cholesterol-containig "black" phospholipid membrane (thickness of ~ 51 ?). The length of acyl tails of membrane phospholipids was shown to play a profound role in the formation of amphotercin and subunit B channels capable of existing in the long open state. The ion-conducting properties of bilayer membranes modified with calixarene C-99 suggest the formation of potential-dependent weakly anion-selective ionophore. The results obtained are a scientific basis for a concept on the molecular mechanisms regulating active transport and reception of neurotransmitters in presynapse . Product Description popup.authors І.О.Трикаш А.С. Тарасенко Базилянська В.Р. Воєйков А.М. Л.М. Яценко Л.О. Касаткіна М.В. Лінецька М.Т. Пархоменко Митрофанова Л.О. Н.В. Крисанова Н.Г. Гіммельрейх Н.Г. Позднякова О.О. Крупко О.Я. Шатурський Р.В. Сівко popup.nrat_date 2020-04-02 Close