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Proceedings of the National Academy of... Aug 1979A synaptosomal ATP-dependent Ca uptake system was reconstituted into artificial vesicles by a cholate dialysis procedure using an 80-fold excess of exogenous...
A synaptosomal ATP-dependent Ca uptake system was reconstituted into artificial vesicles by a cholate dialysis procedure using an 80-fold excess of exogenous phospholipid. Under these conditions, most of these vesicles would be expected to have only one or, at most, a few membrane proteins. The vesicles containing an ATP-dependent Ca transport system were purified from the bulk of the preparation on density gradients by increasing their density by the ATP-dependent intravesicular precipitation of Ca oxalate; a approximately 100-fold purification resulted. The purified Ca-transporting vesicles contained two major protein components, of Mr 94,000 and 140,000 according to sodium dodecyl sulfate gel electrophoresis. These components are believed to be responsible for Ca transport in this synaptosome-derived membrane fraction.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biological Transport, Active; Brain; Calcium; Macromolecular Substances; Membrane Proteins; Molecular Weight; Rats; Synaptosomes
PubMed: 158762
DOI: 10.1073/pnas.76.8.3708 -
European Journal of Pharmacology Dec 2019Inhibiting glutamate release can reduce neuronal excitability and is recognized as a key mechanism of anti-epileptic drugs. In this study, by using isolated nerve...
Inhibiting glutamate release can reduce neuronal excitability and is recognized as a key mechanism of anti-epileptic drugs. In this study, by using isolated nerve terminal (synaptosome) and slice preparations, we investigated the effect of asiatic acid, a triterpene isolated from Centella asiatica with antiepileptic activity, on glutamate release in the hippocampus of rats. In hippocampal synaptosomes, application of asiatic acid resulted in a concentration-dependent inhibition of 4-aminopyridine-evoked glutamate release. This inhibitory action was dependent on extracellular calcium, blocked by inhibiting the vesicular transporter, but was unaffected by inhibiting the glutamate transporter. In addition, asiatic acid decreased the 4-aminopyridine-induced increase in the intraterminal calcium and failed to alter the synaptosomal potential. Furthermore, the asiatic acid-mediated release inhibition was significantly suppressed by the N- and P/Q-type calcium channel inhibitor ω-conotoxin MVIIC or protein kinase C inhibitor GF109203X. Western blotting data in synaptosomes also revealed that asiatic acid reduced 4-aminopyridine-induced phosphorylation of protein kinase C. In hippocampal slices, asiatic acid decreased the frequencies of spontaneous excitatory postsynaptic currents without changing their amplitudes and glutamate-activated currents in CA3 pyramidal neurons. We also observed that asiatic acid significantly suppressed 4-aminopyridine-induced burst firing. These data suggest that, in rat hippocampal nerve terminals, asiatic acid attenuates the calcium influx via N- and P/Q-type calcium channels, subsequently suppressing protein kinase C activity and decreasing glutamate release.
Topics: 4-Aminopyridine; Animals; Calcium; Centella; Glutamic Acid; Hippocampus; Indoles; Male; Maleimides; Pentacyclic Triterpenes; Protein Kinase C; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Synaptosomes; omega-Conotoxins
PubMed: 31706856
DOI: 10.1016/j.ejphar.2019.172781 -
Doklady Akademii Nauk SSSR 1981
Topics: Animals; Cell Membrane; Dopamine; Hypothalamus; In Vitro Techniques; Rats; Synaptosomes; Tyrosine; Tyrosine 3-Monooxygenase
PubMed: 6113923
DOI: No ID Found -
Neurotoxicology and Teratology 1998This study evaluates the potential of two chelators, 1,2-dimethyl-3-hydroxypyridine-4-one (Hdpp) and 1-n-butyl-2-methyl-3-hydroxypyridin-4-one (Hnbp), to modulate...
This study evaluates the potential of two chelators, 1,2-dimethyl-3-hydroxypyridine-4-one (Hdpp) and 1-n-butyl-2-methyl-3-hydroxypyridin-4-one (Hnbp), to modulate cerebral rates of free radical production. The fluorometric assay for 2',7'-dichlorofluorescein, which is formed by oxidation of a nonfluorescent precursor (2',7'-dichlorofluorescein diacetate), was used to assay reactive oxygen species (ROS) production. The chelator Hdpp alone and the aluminum complexes of each chelator, Al (dpp)3 and Al (nbp)3, all inhibited basal rates of generation of ROS within a rat cerebral synaptosomal fraction. In the presence of an iron salt (1 microM FeSO4), a major enhancement of synaptosomal ROS formation was apparent. However, with the addition of an equimolar concentration of Hdpp, Al(dpp)3, or Al(nbp)3, this stimulation was completely abolished. The N-substituted-3-hydroxy-4-pyridinones have been proposed to be of clinical utility for the removal of iron or aluminum from tissues. The clinical potential of this class of chelator may be enhanced by their ability to inhibit iron-related oxidative events.
Topics: Aluminum; Analysis of Variance; Animals; Chelating Agents; Dose-Response Relationship, Drug; Male; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Synaptosomes
PubMed: 9638689
DOI: 10.1016/s0892-0362(97)00103-7 -
The Journal of Biological Chemistry Apr 1977Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi,...
Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.
Topics: Animals; Biological Transport, Active; Brain; Calcimycin; Calcium; Kinetics; Male; Microscopy, Electron; Molecular Weight; Nerve Tissue Proteins; Phosphoproteins; Potassium; Rats; Subcellular Fractions; Synaptosomes; Veratridine
PubMed: 323254
DOI: No ID Found -
Indian Journal of Biochemistry &... Apr 1982
Topics: Animals; Corpus Striatum; Dopamine; Pheniramine; Rats; Structure-Activity Relationship; Synaptosomes
PubMed: 7129508
DOI: No ID Found -
Journal of Neuroscience Research Feb 1990The ability of altered environmental conditions to modulate some properties of synaptosomes has been studied. Incubation conditions used included the presence of methyl...
The ability of altered environmental conditions to modulate some properties of synaptosomes has been studied. Incubation conditions used included the presence of methyl mercury or an organochlorine insecticide: chlordecone. Other adverse chemical conditions during incubation were the absence of calcium salts from the incubation medium or the addition of agents bringing about enhanced oxidative conditions. Synaptosomal parameters studied were the cytosolic level of free, ionic calcium, [Ca2+]i, the extent of depolarization-induced uptake of radioactive calcium, and the permeability of the limiting membrane. In addition, peroxidative activity was estimated by quantitation of thiobarbituric acid-reactive material. All these facets of synaptosomal function were responsive to the presence of these potentially deleterious changes in the incubation medium. While the response of [Ca2+]i was potentially in either direction, all adverse conditions increased synaptosomal permeability as evaluated by leakage of fura-2 into the extracellular compartment. Pretreatment with ganglioside GM1 in some situations or alpha-tocopherol in others could either wholly or partially prevent the onset of such altered synaptosomal characteristics.
Topics: Animals; Calcium; Chlordecone; Insecticides; Male; Methylmercury Compounds; Oxidation-Reduction; Rats; Synaptosomes; Vitamin E
PubMed: 1690820
DOI: 10.1002/jnr.490250211 -
Journal of Neurochemistry Dec 1976
Topics: Amino Acids; Animals; Electric Stimulation; Female; In Vitro Techniques; Limbic System; Microscopy, Electron; Nerve Endings; Oxygen Consumption; Rats; Synaptosomes; Time Factors
PubMed: 1003222
DOI: 10.1111/j.1471-4159.1976.tb02633.x -
Brain Research Dec 1984In nerve terminals, glutamate (Glu) may serve as precursor of the inhibitory neurotransmitter, GABA, and the putative excitatory transmitter, aspartate (Asp), in...
In nerve terminals, glutamate (Glu) may serve as precursor of the inhibitory neurotransmitter, GABA, and the putative excitatory transmitter, aspartate (Asp), in addition to exerting its own excitatory neurotransmitter role in brain. Glu carbon can originate from glucose through glycolysis and the Krebs cycle, from glutamine (Gln) subsequent to uptake, and from proline (Pro) and ornithine (Orn). Orn, but not Glu, is an effective precursor in nerve terminals of Pro, a putative inhibitory neurotransmitter. [3H]Arg can be converted in mouse brain nerve terminals to Orn, which in turn gives rise to Glu, Pro and GABA. In the present study, the conversion subsequent to uptake of labeled Glu, Gln and Pro to other amino acids was studied in unfractionated and subfractionated synaptosomal particles which layered, respectively, on 1.0 M, 1.2 M, 1.3 M and 1.5 M sucrose after centrifugation in a discontinuous gradient (fractions 1-4, respectively). Fraction 1 contained small synaptosomal fragments with vesicles and almost no mitochondria. Fractions 2 and 3 showed numerous normal-appearing mitochondria-containing synaptosomes, and fraction 4 contained large synaptosomes and more free mitochondria than the other fractions. Glu was readily taken up in all fractions and converted to Asp, Gln and GABA, the greatest formation of Asp from Glu occurring in fractions 2 and 3 and of Gln in fraction 4. In contrast, Gln was taken up poorly in fraction 1 and not metabolized, converted extensively to Glu and GABA in fractions 2-4, giving rise only to very small amounts of Asp in fractions 2 and 3. Although Pro was taken up to the greatest extent in fraction 2, it was by far most readily converted to Glu, Gln and GABA in fraction 1, showing only small amounts of Asp formation in fractions 1-3 and none in 4. There was no significant production of Pro from Glu or Gln or of Arg and Orn from any of the 3 precursors studied. The above results suggest that Glu, Gln and Pro may be taken up largely in different classes of synaptosomes which are distributed among the centrifugally separated fractions and which possess differing transport and metabolic characteristics. Determination of glutamate decarboxylase activity (GAD) indicated that GABA-forming nerve terminals were present in all synaptosomal fractions studied. Amino acid determinations by HPLC in the subfractionated synaptosomes showed a similar distribution for Glu, Asp and GABA contents, peaking in fraction 2, and an inverse relationship of the latter 3 with Arg contents.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Amino Acids; Animals; Brain; Brain Chemistry; Cell Fractionation; Glutamate Decarboxylase; Glutamates; Glutamic Acid; Glutamine; Mice; Proline; Synaptosomes
PubMed: 6151865
DOI: 10.1016/0006-8993(84)90295-6 -
Neuroscience Letters Jun 1988The effect of ethanol on synaptosomal free calcium concentration [( Ca2+]i) and 45Ca2+ uptake was studied in vitro. Synaptosomes were prepared from forebrains of adult...
The effect of ethanol on synaptosomal free calcium concentration [( Ca2+]i) and 45Ca2+ uptake was studied in vitro. Synaptosomes were prepared from forebrains of adult rats and incubated in the presence of ethanol 50-500 mM. [Ca2+]i determined by means of the Ca2+-sensitive dye, fura-2, was increased by ethanol in polarized and depolarized synaptosomes, whereas 45Ca2+ uptake was decreased. The results suggest that ethanol may influence neuronal calcium homeostasis by increasing rather than decreasing [Ca2+]i.
Topics: Animals; Calcium; Ethanol; Male; Rats; Rats, Inbred Strains; Synaptosomes
PubMed: 3393294
DOI: 10.1016/0304-3940(88)90375-8