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PubMed1980

[Osmotic properties of heavy and light synaptosomes of the rat cerebral cortex].

A comparison has been carried out of osmotic properties of light and heavy synaptosomal fractions and a mitochondrial fraction from the rat brain cortex. A critical change is the rate of swelling at a definite time, allowing to subdivide the process into two phases, was shown. Throughout the swelling, its rate for heavy synaptosomes and mitochondria was much higher than that for the light ones. According to the initial rate of swelling in isoosmotic glycerol, the fractions examined can be arranged in the following order: mitochondria < light synaptosomes < heavy synaptosomes. Both total, light and heavy synaptosomes reveal the properties of ideal osmometers. The osmotic properties of synaptosomes and mitochondria are largely dependent on pH of the medium: with a shift towards acidic values of pH the swelling rate decreases to increase at more alkaline values. Especially well-pronounced changes occur at pH 6.8 and pH 7.3.

Animals↗

PubMed1980

Inhibition of synaptosomal calcium uptake by ethanol.

In vitro addition of ethanol or pentobarbital to synaptosomes isolated from rat or mouse brain inhibited the depolarization-dependent uptake of calcium without affecting uptake under nondepolarizing conditions. Ethanol inhibited the uptake in a concentration-dependent manner over the range of 45 to 720 mM. Analysis of the effects of ethanol and pentobarbital on calcium uptake at different temperatures indicated that the drugs did not change the activation energy of the process but shifted the Arrhenius curves toward higher temperatures. Synaptosomes isolated from mice chronically ingesting ethanol were found to be tolerant to the inhibitory effects of ethanol and pentobarbital but not tolerant to the inhibitory effects of acetaldehyde. Synaptosomes from ethanol tolerant-dependent mice accumulated less calcium in the absence of ethanol than did synaptosomes from control mice. This depression of uptake was reversed by in vitro exposure of the synaptosomes to a low concentration of ethanol, suggesting that it represents a biochemical response to withdrawal of alcohol. A single acute injection of ethanol 1 hr before sacrifice did not alter the calcium uptake activity or the drug sensitivity of the synaptosomes. These results suggest that the known inhibitory effects of ethanol and pentobarbital on the stimulated release of neurotransmitters and the development of tolerance of these effects may be mediated by the inhibition of the depolarization-dependent influx of calcium by these drugs.

Alcoholism↗

PubMed1980

Acute and chronic effects of barbiturates on depolarization-induced calcium influx into synaptosomes from rat brain regions.

Male Sprague-Dawley rats (250--300 g) were randomly divided into three experimental groups: control, acute (received a single dose of 60 mg/kg of phenobarbital i.p.) and tolerant (received dietary phenobarbital, 2.5 mg/kg of milled Purina Lab Chow, for 13 days). Cerebral cortex (CT), cerebellum (CB), brain stem (BS), striatum (ST), hypothalamus (HT), and mid-brain (MB) were isolated, and the inhibitory effects of barbiturates on KCl-induced 45Ca++ influx into synaptosomes from each of these brain regions isolated from each experimental group were examined. KCl-induced 45Ca++ influx into CT, CB and BS synaptosomes from control animals was significantly depressed (39, 73 and 77% respectively) by an in vitro pentobarbital (0.3 mM) challenge. No significant depression of 45Ca++ influx was produced by pentobarbital in control synaptosomes isolated from ST, HT or MB. KCl-induced 45Ca++ influx into synaptosomes from tolerant rats was not significantly depressed by the in vitro pentobarbital challenge in any brain area. The results suggest that 45Ca++ influx into synaptosomes from CT, CB and BS is more susceptible than ST, HT and MB to inhibition by barbiturates. In addition, chronic administration of phenobarbital resulted in the adaptation of CT, CB and BS synaptosomes to the inhibitory effects of in vitro pentobarbital. This adaptation occurred in the same time frame as did the development of behavioral tolerance.

Animals↗

PubMed1997

Enhanced dopamine release and phosphorylation of synapsin I and neuromodulin in striatal synaptosomes after repeated amphetamine.

Repeated, intermittent treatment of rats with amphetamine followed by a withdrawal period leads to an enhancement in amphetamine-induced dopamine release. We previously reported an increased stoichiometry of site 3-phospho-synapsin I and increased levels of phospho-Ser41-neuromodulin in striatum after repeated amphetamine. In this study, we examined whether the enhanced amphetamine-induced dopamine release and increased levels of these phosphoproteins would be detected in synaptosomes from rats pretreated and withdrawn from repeated amphetamine. Enhanced amphetamine-induced dopamine release was detected in striatal synaptosomes from rats treated with repeated amphetamine compared with controls. The enhanced dopamine release was Ca++ dependent. State-specific antibodies were used to measure the levels of site 3-phospho-synapsin I, phosphorylated by CaM kinase II, and phospho-Ser41-neuromodulin, phosphorylated by protein kinase C, in incubated striatal S1 fractions and synaptosomes. The levels of site 3-phospho-synapsin I and phospho-Ser41-neuromodulin were increased by 40% and 30%, respectively, in amphetamine-pretreated rats compared with controls. Total neuromodulin and synapsin I was not altered. There was a significant 26% increase in CaM kinase II activity in the synaptosomes from amphetamine-pretreated rats but no change in content. No change in protein kinase C activity or content of the alpha-isozyme was detected after repeated amphetamine. Our results demonstrate that the enhanced amphetamine-induced dopamine release and occurring after repeated amphetamine can be detected in synaptosome preparations. Repeated amphetamine leads to alterations in phosphorylation/dephosphorylation activities that can be detected in the incubated synaptosomes. Because the enhanced amphetamine-induced dopamine release after repeated amphetamine appears to be Ca++ sensitive, it is possible that the altered phosphorylation systems, and perhaps site 3-phospho-synapsin I and phospho-Ser41-neuromodulin, play a role in the enhanced dopamine release.

Amphetamine↗

PubMed1968

The uptake of [14C] choline into synaptosomes in vitro.

1. The uptake of [(14)C]choline into synaptosomes in vitro was investigated by a gel-filtration method. Synaptosomes incubated in a medium fortified with glucose and succinate rapidly take up [(14)C]choline. 2. A substantial proportion of the radioactivity taken up can be released by osmotic shock, and is recoverable as choline on a thin-layer chromatogram. This suggests that choline is taken up across the limiting membrane into the cytoplasmic compartment of the synaptosome. 3. The concentration of choline in the synaptosome has a dependence on the external concentration of choline that is similar to that in erythrocytes and mouse cerebral-cortex slices. The choline influx has two components, one that is linear and one that is saturable with increasing choline concentration. 4. Omission of Na(+) from the incubation medium, or addition of 100mm-K(+), inhibits choline uptake. Hemicholinium no. 3 is a powerful inhibitor of the choline uptake. 5. The similarity of the choline-uptake process in synaptosomes to that in erythrocytes and cortex slices indicates that the synaptosome limiting membrane is functionally competent in this respect.

Animals↗

PubMed1985

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin.

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Animals↗

PubMed1983

Synaptosomal T3 binding sites in rat brain: their localization on synaptic membrane and regional distribution.

Previous studies have shown the existence of specific high affinity T3 binding sites in rat cerebrocortical synaptosomes. In this study, to define the localization of the binding sites, T3 binding to disrupted synaptic membrane fractions was compared with the binding to intact synaptosomes obtained from the rat cerebral cortex. Scatchard analysis revealed two orders of T3 binding sites with almost identical apparent dissociation constant (Kd) in synaptosomes and synaptic membrane fractions. However, the maximal binding capacity (MBC) of the higher affinity binding sites for synaptic membrane fractions was significantly greater than that for intact synaptosomes (4.2 +/- 0.2 vs 3.0 +/- 0.3 pg T3/mg protein). Regional distribution of the synaptosomal T3 binding sites in various areas of the rat brain was also studied. The Kd values were not significantly different in discrete brain regions. On the other hand, the MBCs of the higher affinity binding sites were greater in the cerebral cortex and hypothalamus (72.8 +/- 1.4 and 64.8 +/- 9.5 pg T3/g tissue) than in the cerebellum (22.1 +/- 1.6 pg T3/g tissue). Similar difference was also observed in the lower affinity sites. These results indicate that the specific T3 binding sites in brain synaptosomes are localized mainly on synaptic membranes and that the MBC is different in discrete brain regions.

Animals↗

PubMed2003

Antagonism of botulinum toxin type A-induced cleavage of SNAP-25 in rat cerebral synaptosome by toosendanin.

Toosendanin (TSN), a triterpenoid derivative extracted from Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study is designed to explore its antibotulismic mechanism by Western blotting. The results showed that TSN incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes, but made the synaptosomes completely resistant to botulinum neurotoxin A (BoNT/A)-mediated cleavage of SNAP-25. After binding of BoNT/A to synaptosomes, TSN still partially antagonized the toxin-mediated cleavage of SNAP-25. However, TSN-incubated synaptosomal membrane fraction did not resist the cleavage of SNAP-25 by the light chain of BoNT/A. It is suggested that the antibotulismic effect of TSN results from blocking the toxin's approach to its enzymatic substrate.

Animals↗

PubMed1998

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens.

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline-soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity-purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen-driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1980

Sodium dependence of GABA transport in rat hypothalamic synaptosomes.

The sodium dependence of gamma-aminobutyric acid (GABA) uptake has been studied in rat hypothalamic synaptosomes, and the results compared to previous studies in cortical synaptosomes. Initial velocity of GABA uptake was measured as a function of both GABA and sodium concentration, and these data were fitted to the rate equation for each of several plausible models. The minimal best-fit model was found to be identical to that for cortical synaptosomes. However, the constants which quantitate the model were found to differ for hypothalamus and cortex. As a result, uptake at any combination of [Na] and [G] in hypothalamic synaptosomes is approximately double that in cortical synaptosomes. The rate equation for the minimal best-fit model was utilized to define and compute certain parameters which are useful in comparing transport mechanisms (Vmax, Va, Kt, Jm, and k Na). In all cases, differences were found between hypothalamus and cortex.

Animals↗

PubMed1982

Effect of phospholipase A2 on calcium transport in brain synaptosomes.

Pretreatment of isolated brain synaptic endings with commercial phospholipase A2 isolated from venom of Apis mellifera, followed by a BSA washing, selectively inhibited the depolarization-dependent portion of 45Ca uptake. Phospholipase A2 initially caused an increase of synaptosome respiration and subsequently inhibited their oxygen uptake, but this effect was completely abolished in BSA-containing media. The classical uncoupler of oxidative phosphorylation, DNP, produced release of 45Ca or [3H]GABA from superfused synaptosomes previously loaded with radioactive calcium or GABA. The treatment of synaptosomes with phospholipase A2 had no effect on the spontaneous efflux of 45Ca or [3H]GABA. However, depolarization-dependent release of [3H]GABA from synaptosomes treated with phospholipase A2 was significantly inhibited. We suggest that the inhibition of depolarization-dependent influx of 45Ca into synaptosomes treated with phospholipase A2 may be attributed to the lesion of the specific function of plasma membrane rather than to the impairment of the calcium-sequestrating function of intraterminal structures.

2,4-Dinitrophenol↗

PubMed1990

Prevention of chemically induced synaptosomal changes.

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.

Animals↗

PubMed1998

The protective effect of vitamin E, idebenone and reduced glutathione on free radical mediated injury in rat brain synaptosomes.

In the present study the effect of ascorbate (0.8 mM)/iron (2.5 microM) on lipid and protein oxidation, in Synaptosomes isolated from rat brain cortex, was evaluated. Vitamin E, idebenone and reduced glutathione were used as free radicals scavengers, in order to analyze the mechanism involved in ascorbate/iron-induced oxidative stress. An increased formation of reactive oxygen species (ROS) in the cytosol and in the mitochondria was observed, in ascorbate/iron treated synaptosomes. Idebenone (50 microM) prevented the increased formation of ROS in both synaptosomal compartments, vitamin E (150 microM) protected partially this formation in mitochondria, whereas reduced glutathione (250 microM) (GSH) was ineffective. After ascorbate/iron treatment an increase in lipid peroxidation occurred as compared to control, which was completely inhibited by idebenone. A decrease in protein-SH content was also observed, and it was prevented by Vitamin E, idebenone and GSH. When synaptosomes were treated with ascorbate/iron the levels of GSH decreased, and the levels of oxidized glutathione (GSSG) increased as compared to controls under these conditions. Glutathione peroxidase activity was unchanged, whereas an inhibition of glutathione reductase activity was observed. These data suggest that the increased formation of free radicals in synaptosomes leads to lipid and protein oxidation, the role of the endogenous GSH being essential to protect protein thiol-groups against oxidative damage in order to maintain enzyme activity.

Adenine Nucleotides↗

PubMed1988

Effects of lithium on synaptosomal Ca2+ fluxes.

Lithium is the primary treatment for mania, and it has been employed as a therapeutic agent in the treatment of a number of other conditions. In spite of its widespread clinical use, the specific mechanism by which lithium acts is still not known. Because lithium bears some chemical resemblance to Ca2+ and because it has been found to interfere with many Ca2+-dependent processes, we investigated the possibility that lithium can alter intracellular Ca2+ homeostasis by an effect on Ca2+ fluxes. When added in vitro to synaptosomes prepared from rat forebrains, lithium had no effect on 45Ca2+ influx mediated by fast or slow phase depolarization-dependent Ca2+ channels or by Na+/Ca2+ exchange. In vitro treatment with lithium also had no effect on ATP-dependent Ca2+ sequestration by mitochondria or synaptosomal endoplasmic reticulum. In contrast, 45Ca2+ influx in synaptosomes prepared from rats treated chronically with lithium was increased significantly relative to controls. The fact that chronic in vivo treatment with lithium affected synaptosomal Ca2+ flux whereas in vitro treatment did not is consistent with the time course of therapeutic effectiveness in man. The results suggest that lithium does not directly interact with synaptosomal Ca2+ flux, but rather may influence Ca2+ flux through an indirect mechanism following chronic treatment.

Animals↗

PubMed1978

Protein synthesis by synaptosomes from rat brain: the effect of centrifugal forces.

1. Synaptosomal protein synthesis has been found to differ depending on whether synaptosomes are recovered from the sucrose gradients by centrifuging at 20,000 g or at 150,000 g. 2. The fraction obtained at 20,000 g incorporated 60% more amino acid into proteins after 30 min of incubation than the one obtained in the same period of time at 150,000 g. 3. The respiratory capacity of synaptosomes obtained at 20,000 g in the presence or absence of glucose as substrate, is also better preserved than that of those obtained at 150,000 g. 4. It is shown that the yield, enzymic content, and nucleic acid composition is the same in both fractions. 5. These results cannot be explained by the presence of inhibitory factors in the 150,000 g fraction or by the presence of better preserved free mitochondria in the 20,000 g fraction. 6. Our findings are best explained by postulating that the synaptosomal metabolic activity is damaged when synaptosomes are pelleted at too high a speed.

Acetylcholinesterase↗

PubMed1984

The ouabain-sensitive component of synaptosomal respiration.

The relationship of oxygen consumption with the activity of the synaptic Na-K-pump was investigated in rat cortical synaptosomes. Changes in oxygen consumption were monitored during the restitution of physiological ionic equilibrium in incubated synaptosomes and under the effects of ouabain and protoveratrine. The ouabain-sensitive component of synaptosomal respiration was measured and correlated with earlier data on synaptosomal K+-uptake. This ouabain-sensitive component was shown to reflect the activity of the Na-K-pump. Our results thus provide a means for the continuous monitoring of ion-pump activity in intact synaptosomes.

Animals↗

PubMed1987

Laser light scattering determination of size and dispersity of synaptosomes and synaptic vesicles isolated from squid (Loligo pealei) optic lobes.

Quasi-elastic laser light scattering has been used to investigate the size and dispersity of synaptosomes and synaptic vesicles isolated from optic lobes of the squid Loligo pealei. Synaptosomal fractions were highly polydisperse (mu2/gamma -2 = 0.5) and the mean diameter (-d) ranged from 0.5-2.0 microns. Size distribution histograms yielded two major components - smaller particles (-d approximately 300-700 nm) and a larger group of particles (-d approximately 1,500-5,000 nm). The heterogeneity of the synaptosomal particles detected in solution is in agreement with published data obtained using electron microscopy. Purified synaptic vesicle fractions also yielded complex particle size distribution data. A component with a mean diameter in the range 150-250 nm was detected, though a smaller particle (-d approximately 40-110 nm) dominated the scattering signal. This smaller particle closely resembles in size the electron lucent vesicles seen in the majority of squid optic lobe nerve terminals when examined by electron microscopy. Osmotically-induced shrinkage and swelling of the synaptosomes was detected. Depolarization by veratridine (1.0 x 10(-4) M) did not result in a detectable change in the size of synaptosomal particles.

Animals↗

PubMed1977

Substance P in rat brain synaptosomes.

1. Rat brain synaptosomes were incubated under different conditions to study the release of substance P (SP). 2. Potassium ions and electrical field stimulation induced a loss of SP from synaptosomes. The release of SP by potassium in high concentrations (23.8 mM) was shown to be calcium dependent. 3. Substance P was retained in synaptosomes during incubation in 0.32 M sucrose at +4 degrees C up to 120 min. During incubation at 30 degrees C the SP content fell initially (30 min) but was gradually restored (120 min). 4. If these pre-incubated synaptosomes were reincubated for 45 min at 30 degrees C in potassium free Krebs-Ringer-phosphate buffer a further rise in their SP content occurred which was taken as indication that SP is being synthesized in synaptosomes. 5. The newly synthesized SP is presumably stored by binding to phosphatidyl serine until a sudden release is initiated by depolarization.

Animals↗