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PubMed1985

In vivo effects of aminooxyacetic acid and valproic acid on nerve terminal (synaptosomal) GABA levels in discrete brain areas of the rat. Correlation to pharmacological activities.

A newly developed synaptosomal model was used to evaluate the in vivo effects of the GABA-elevating drugs aminooxyacetic acid (AOAA, 30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) on GABA levels in nerve endings of 11 brain regions in rats as a function of time after administration. The data obtained were compared with the magnitude and time course of the effects of both drugs in rats on body temperature, pain response and against seizures induced by electroshock, pentylenetetrazol and 3-mercaptopropionic acid. Following AOAA, maximum increases in synaptosomal GABA levels of brain regions were observed 6 hr after administration. At this time, GABA was significantly elevated up to 300% over control values in synaptosomal fractions from all 11 regions. However, the hypothermic and antinociceptive effects of the drug as well as its anticonvulsant action against electroshock and pentylenetetrazol induced seizures were maximal 1 hr after injection and had vanished after 6 hr, i.e. at the time of maximum GABA increases in synaptosomes. The only pharmacological effect of AOAA which paralleled the time course of the synaptosomal GABA elevation was the attenuation of seizures induced by 3-mercaptopropionic acid. Following VPA, the effect on synaptosomal GABA levels was much more rapid in onset and significant increases were already determined 5 to 30 min after administration. Significant increases of up to 80% over control values were found in synaptosomal fractions from olfactory bulb, frontal cortex, hippocampus, hypothalamus, tectum, substantia nigra and cerebellum. In contrast to AOAA, the time course of the synaptosomal GABA increases, at least in some regions, was similar to the time course of VPA's antinociceptice effects and its anticonvulsant effects in the three seizure models studied. The data may suggest that AOAA and VPA increase different pools of GABA within nerve terminals, only one of which is involved in GABA-mediated neurotransmission.

Acetates↗

PubMed1980

Release of [14C]adenosine derivatives from superfused synaptosome preparations. Effects of depolarizing agents and metabolic inhibitors.

Adenine 5'-mononucleotides of synaptosome beds from guinea pig neocortex were labelled by incubation with [8-(14)C]adenosine, and excess adenosine was then removed with precursor-free medium. During continuous superfusion, labelled adenosine derivatives were released at a rate of about 0.5% of the synaptosome 14C/min and this rate was increased 2.5-fold by depolarization with 33 mM K+. Some depolarizing agents and metabolic inhibitors were examined for action on the release of [14C)-adenosine derivatives from the synaptosome preparations, and also on the rate of lactate production by these preparations, both before and during K + depolarization. The synaptosome content of adenine 5'-mononucleotides following exposure of the synaptosome beds to these compounds was also estimated. Ouabain, 0.1 mM, brought about an increase in 14C-labelled compounds output, but it caused little alteration in lactate output and some decrease in the adenylate energy charge of the synaptosomes. Veratridine, 80 microM, increased markedly both the output of radioactivity and the lactate production. Tetrodotoxin, 1 microM, when added together with veratridine, completely abolished the effect of veratridine on the efflux of [14C]adenosine derivatives, but this was not associated with a complete blockade of the output of lactate. Sodium cyanide, 2.5 mM, FCCP, 6 muM together with iodoacetate, 2.5 mM, caused an increase in the output of 14C-labelled compounds, and a decrease in the adenylate energy charge. The production of lactate was also increased by sodium cyanide and FCCP, but it was completely inhibited by iodoactate. Oligomycin, 4.65 microM, and amytal, 1 mM, added in the incubation medium before labelling the synaptosome beds with [8-(14)C]adenosine, did not affect very much the output of [14C]adenosine derivatives, while the output of lactate increased independently of the depolarization with 33 mM K+. The release of adenosine derivatives from superfused synaptosome beds induced by depolarizing agents or metabolic inhibitors did not seem to be due either to an effect of membrane permeability changes that follow a decrease of ATP supply, or to an increased metabolic rate occurring during nerve ending stimulation. It is concluded that this release of adenosine derivatives appeared to be a process triggered primarily by the influx of Na+ and the increased intrasynaptosomal calcium, and closely related to the process of neurotransmission.

Adenine Nucleotides↗

PubMed1988

Calcitonin gene-related peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn.

Effects of synthetic human calcitonin gene-related peptide (hCGRP) on Ca2+ mobilization in crude synaptosomes prepared from rat spinal dorsal horn were studied using 45Ca2+ as a tracer. hCGRP promoted the influx of 45Ca2+ into synaptosomes of the spinal dorsal horn, in a dose dependent manner (at dose ranges from 10(-9) to 10(-6) M). The maximum effect of hCGRP on 45Ca2+ influx was achieved 5 min after addition of the peptide and was significantly inhibited by nifedipine. hCGRP did not alter the influx of 45Ca2+ into synaptosomes of the rat cerebral cortex, a brain area with reportedly few CGRP binding sites. In measurements of the 45Ca2+ efflux from synaptosomes of the spinal dorsal horn, 10(-7) M of hCGRP had no effect on the efflux of 45Ca2+ from the resting synaptosomes, but there was significant inhibitory effect on synaptosomes depolarized by 40 mM K+. The inhibitory effect of hCGRP on the efflux of 45Ca2+ was completely abolished by lowering the incubation temperature of the medium to 0 degrees C. The inhibitory effect of hCGRP on the efflux of 45Ca2+ was not observed in synaptosomes of the cerebral cortex. These findings suggest that hCGRP increases the levels of Ca2+ level in synaptosomes of the spinal dorsal horn, through interactions with its own receptors and the release of neurotransmitters from the nerve terminals is thus enhanced.

Animals↗

PubMed1982

Drug-induced release of biogenic amines from synaptosomes and blood platelets of guinea-pigs.

Synaptosomes from guinea-pig brain were compared with blood platelets (partly from previous experiments) regarding the action of imipramine, the benzoquinolizine derivative Ro 4-1284, tyramine and p-chlormethamphetamine (PCMA) on the contents of stored radio-labelled 5-hydroxytryptamine (5HT), dopamine (DA) and noradrenaline (NA). Normal and reserpinized preparations were used in order to differentiate between granular and extragranular (reserpine-resistant) sites. Imipramine, in concentrations greater than those inhibiting 5HT- and NA-uptake released the three amines from granular and extragranular sites and showed the same order of potency in synaptosomes as in platelets. The drug Ro 4-1284 acted on the granular amines only, but its action on NA was less potent in synaptosomes than in platelets. Tyramine and PCMA also caused an exclusive release of granular amines in synaptosomes, whereas in platelets the drugs released extragranular 5HT (and DA) as well as the granular amines. Tyramine was more potent in synaptosomes than in platelets, and PCMA showed a preferential effect on 5HT in synaptosomes but not in platelets. It is concluded that, regarding the action of monoamine-releasing drugs, platelets are only partial models for synaptosomes. However, they may be superior to synaptosomes in predicting the pattern of amine release in brain in vivo as seen with Ro 4-1284.

2H-Benzo(a)quinolizin-2-ol, 2-Ethyl-1,3,4,6,7,11b-↗

PubMed1995

Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-CgTx GVIA.

We describe a magnetophoretic method for the affinity purification of synaptosomes expressing omega-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of omega-CgTx GVIA which retains its ability to displace [125I] omega-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between omega-CgTx GVIA and the biotin:streptavidin bead complex is sufficiently long to allow flexibility of the toxin to bind to its receptor on synaptosomes. We have used this ligand successfully to isolate deaggregated synaptosomes from the parent fractions of chicken forebrain and rat cortex. In the chicken synaptosome parent fraction, omega-CgTx GVIA (1 nM-1 microM) produced a concentration-dependent block of the KCl-induced intracellular free Ca2+, [Ca2+]i, elevation with an IC50 of 28 nM. After affinity magnetophoresis no increase in [Ca2+]i elevation was observed in either the bound or unbound fractions. In the rat synaptosome parent fraction, the KCl-induced increase in free intracellular Ca2+ ([Ca2+]i) elevation was partially blocked by omega-CgTx GVIA (17 +/- 2% / 1 microM) and to a greater extent by omega-Aga IVA (55 +/- / 1 microM): a combination of the two toxins was additive (72 +/- 4% / 1 microM). The block obtained by omega-CgTx GVIA (1 microM) in the unbound fraction was reduced to 3 +/- 2%, whereas that by omega-Aga IVA (1 microM) increased to 82 +/- 3%. The block obtained by a combination of both toxins (83 +/- 2%) was the same as that with omega-Aga IVA alone (82 +/- 3%). No increase in free [Ca2+]i elevation was observed in the bound fraction although single synaptosome-like structures, displaying synaptophysin immunoreactivity, were detected on the beads. We conclude that omega-CgTx GVIA-sensitive N-type calcium channels are present on all chicken forebrain synaptosomes but only a subset of rat cortical synaptosomes.

Animals↗

PubMed1983

Effect of toxaphene on the binding of 3H-labeled ouabain and dopamine to rat brain synaptosomes.

The effects of toxaphene, a chlorinated hydrocarbon pesticide, on the binding of ouabain and dopamine to rat brain synaptosomes enriched with Na+-K+ ATPase were investigated. For in vitro assessment of the effects of toxaphene, the synaptosomes prepared from normal rats were used. For in vivo effects the rats were fed on 0, 50, 100, 150 and 200 ppm toxaphene mixed in their daily ration for 8 weeks. At the end of treatment the rats were killed and synaptosomes were prepared. Toxaphene inhibited Na+-K+ and Mg2+ ATPases of synaptosomes in vitro and the inhibition was significant and concentration-dependent. The IC50 values were about 30 and 12 microM toxaphene for Na+-K+ and Mg2+ ATPases, respectively. However, much higher concentrations of toxaphene were required to inhibit the binding of [3H]ouabain and [3H]dopamine to synaptosomes. A 50% inhibition of ouabain and dopamine binding was obtained at 150 and 200 microM of toxaphene. The enzyme activities of synaptosomes in toxaphene-pretreated rats were decreased significantly. However, a dose-dependent decrease was not observed. The rats receiving dosages of 100 ppm and above showed a 30-40% decrease in enzyme activities. The binding of ouabain and dopamine to synaptosomes of toxaphene-pretreated rats showed no significant changes as compared to controls. The present in vitro results suggest that toxaphene may be an effective inhibitor of ATPases with substantial effects on the binding of ouabain and dopamine to rat brain synaptosomes. However, data obtained through in vivo studies do not support this contention. The reason for this discrepancy may be that the toxaphene is being rapidly metabolized or might not have reached the site of action.

Adenosine Triphosphatases↗

PubMedDate not supplied

Chronic ethanol treatment alters omega-conotoxin and Bay K 8644 sensitive calcium channels in rat striatal synaptosomes.

Two groups of adult Sprague-Dawley rats were maintained on a nutritionally complete liquid diet. In one group, 37% of the calories normally provided by dextrin were replaced with ethanol. Animals were maintained on this diet for eight weeks. The addition of ethanol (200 mM) in vitro significantly inhibited both calcium influx and dopamine release from control synaptosomes but did not alter calcium influx or dopamine release from synaptosomes isolated from ethanol-treated rats. The dihydropyridine calcium channel agonist Bay K 8644 (1 nM) significantly increased both calcium entry and dopamine release from control synaptosomes depolarized with 15 mM KCl. Bay K 8644 (1 nM) had no significant effect on either calcium entry or dopamine release in synaptosomes isolated from ethanol-treated animals. This loss of functional effect was accompanied by a slight (15%) but statistically insignificant increase in the binding of 3H-nitrendipine to striatal membranes from ethanol-treated rats as compared to control. The calcium-channel blocker, omega-conotoxin (500 nM) had no effect on voltage-dependent calcium uptake into synaptosomes prepared from control or ethanol-treated rats. Conotoxin (500 nM) inhibited the voltage-dependent release of endogenous dopamine from synaptosomes isolated from both groups by 36-44%. Ethanol (200 mM) added in vitro to control synaptosomes did not alter conotoxin's inhibition of dopamine release but completely abolished the omega-conotoxin-induced inhibition of dopamine release in synaptosomes isolated from ethanol-treated animals. These results suggest that DHP-sensitive and omega-conotoxin-sensitive calcium channels in rat brain respond differentially to chronic exposure to ethanol.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2001

The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium, nitric oxide synthase, mitochondria, and phospholipase A.

This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2',7'-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca(2+)]i as measured with Fura-2. Blockade of voltage-dependent Ca(2+) channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca(2+)-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.

Alkanes↗

PubMed2000

Synaptosomes isolated from Goto-Kakizaki diabetic rat brain exhibit increased resistance to oxidative stress: role of vitamin E.

Increased oxidative stress is believed to be an important factor in the development of diabetic complications. In this study, the effect of diabetes on the susceptibility of synaptosomes to oxidative stress, induced by the oxidizing system ascorbate/Fe2+, on the activity of antioxidant enzymes and on the levels of glutathione and vitamin E was investigated. Synaptosomes were isolated from brain of 29-weeks-old Goto-Kakizaki (GK) rats, a model of non-insulin dependent diabetes mellitus and from normal Wistar rats. Synaptosomes isolated from GK rats displayed a lower susceptibility to lipid peroxidation, as assessed by quantifying thiobarbituric acid reactive substances (TBARS), than normal rats (5.33 +/- 0.79 and 7.58 +/- 0.7 nmol TBARS/mg protein, respectively). In the absence of oxidants, no significant differences were found between the levels of peroxidation in synaptosomes of diabetic or control rats. Superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase activities were unaltered in the brain of diabetic rats. There were no statistically significant differences in fatty acid composition of total lipids and reduced glutathione levels in synaptosomes of diabetic and control rats. The decreased susceptibility to membrane lipid peroxidation of diabetic rats synaptosomes correlated with a 1.3-fold increase in synaptosomal vitamin E levels. Vitamin E levels in plasma were also higher in diabetic rats (21.32 micromol/l) as compared to normal rats (15.13 micromol/l). We conclude that the increased resistance to lipid peroxidation in GK rat brain synaptosomes may be due to the increased vitamin E content, suggesting that diabetic animals might develop enhanced defense systems against brain oxidative stress.

Animals↗

PubMed1998

Influence of probes for calcium-calmodulin and protein kinase C signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and leukocyte membranes.

The influence of selected inhibitors of calcium signalling on the plasma membrane Ca2+-ATPase activity of rat synaptosomes and peritoneal leukocyte membranes was studied. The calmodulin inhibitor calmidazolium was an efficient inhibitor (50%) of the synaptosomal Ca2+-ATPase activity in a manner competitive with phosphatidylserine. The inhibition by CGS 9343B (30%) was not counteracted by phosphatidylserine. The intracellular calcium antagonist TMB-8 and the protein kinase inhibitor staurosporine and the derivatives CGP 41251 and CGP 42700 hardly affected the synaptosomal Ca2+-ATPase activity. The flavonoid quercetin was a more effective inhibitor of the ATPase activity of synaptosomal than of leukocyte membranes. Phloretin, at relatively high concentrations, caused only a modest inhibition of synaptosomes. The protein kinase C inhibitor sphingosine was a weak inhibitor of the synaptosomal but an effective inhibitor of the leukocyte membrane Ca2+-ATPase activity. The antineoplastic ether phospholipids BM 41.440 (ilmofosine) and ET-18-OCH3 (edelfosine) effectively inhibited the leukocyte membranes whereas the ATPase activity of synaptosomes was significantly increased by 20 microM and slightly inhibited by higher concentrations of these agents. The analogue hexadecylphosphocholine (miltefosine) did not affect the ATPase activity of the synaptosomes and only inhibited that of the leukocyte membranes at concentrations above 20 microM. These results show that several test substances of current interest affect the activity of the plasma membrane Ca2+-ATPase. The effects depend on the origin of the membranes. The investigation does not permit a distinction between direct effects on the enzyme and an interference with its membrane environment although the latter is indicated for the ether phospholipids.

Animals↗

PubMed1998

Increase of the intracellular Ca2+ concentration mediated by transport of glutamate into rat hippocampal synaptosomes: characterization of the activated voltage sensitive Ca2+ channels.

The changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate and D-aspartate into rat hippocampal synaptosomes was studied. Glutamate increased the [Ca2+]i in a dose-dependent manner with an EC50 of 1.87 microM and a maximal increase of 31.5 +/- 0.9 nM. We also observed that stimulation of the synaptosomes with 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 100 microM kainate, or 100 microM D-aspartate increased the synaptosomal [Ca2+]i. The effect of either of these non-NMDA receptor agonists and of D-aspartate was additive, suggesting the activation of two different components (the ionotropic non-NMDA receptors or the glutamate transporters). Stimulation of synaptosomes with 100 microM glutamate increased the [Ca2+]i and prevented the effect of either non-NMDA receptor agonists and the effect of D-aspartate. We also observed that incubation of the synaptosomes with D-aspartate induced the Ca(2+)-independent release of glutamate, possibly through the reversal of the glutamate carrier. The aim of incubating the synaptosomes with D-aspartate was to avoid undesirable secondary activation of glutamate receptors. After incubating the synaptosomes with 100 microM D-aspartate (10 min at 37 degrees C), the subsequent stimulation with D-aspartate increased the [Ca2+]i due to glutamate transport. This increase in [Ca2+]i induced by 100 microM D-aspartate was insensitive to 1 microM nitrendipine, but was inhibited by about 50% by the presence of both 500 nM omega-CgTx GVIA and 100 nM omega-Aga IVA or by 500 nM omega-CgTx MVIIC. We clearly identified two different processes by which glutamate increased the [Ca2+]i in rat hippocampal synaptosomes: activation of non-NMDA receptors and activation of the glutamate transporters. We also characterized the voltage sensitive Ca2+ channels (VSCC) activated as a consequence of the glutamate transport, and determined that class B (N-type) and class A (P or Q-type) Ca2+ channels were responsible for about 50% of the signal.

ATP-Binding Cassette Transporters↗

PubMed1999

Distribution of synaptosomal-associated protein 25 in nerve growth cones and reduction of neurite outgrowth by botulinum neurotoxin A without altering growth cone morphology in dorsal root ganglion neurons and PC-12 cells.

Synaptosomal-associated protein 25 has been regarded as one of the target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptors essential for exocytosis of vesicles in synapses. We have previously reported that cleavage of syntaxin, which is another target-associated soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, with botulinum neurotoxin C1 resulted in inhibition of neurite extension and morphological changes including growth cone collapse and large vacuole formation. As an attempt to explore the mechanism of growth cone extension, we examined the ultrastructural localization of synaptosomal-associated protein 25 in growth cones with or without treatment of botulinum neurotoxin A, which cleaves synaptosomal-associated protein 25. In dorsal root ganglion neurons, light microscopy demonstrated synaptosomal-associated protein 25 immunoreactivity throughout the neurons, including the cell bodies, neurites and growth cones. Using electron microscopy, gold signals immunoreactive for synaptosomal-associated protein 25 were identified diffusely in the cytoplasm of the growth cones. In contrast, in PC-12 cells, a large number of gold signals were localized on the plasma membranes. High levels of signal were also found in the cytoplasm in the central region of the growth cones. We also confirmed that botulinum neurotoxin A treatment reduced neurite extension by about 50%. However, both in dorsal root ganglion neurons and in PC-12 cells we found no differences in the ultrastructure nor in the localization of synaptosomal-associated protein 25 between growth cones with and without toxin treatment. These results indicate that cleavage of synaptosomal-associated protein 25 inhibits growth cone extension in a manner different than that of syntaxin cleavage. The results of this study suggest the possibility that synaptosomal-associated protein 25 is involved in growth cone extension through a process independent of vesicle fusion.

Animals↗

PubMed1998

Cryopreservation of rat cortical synaptosomes and analysis of glucose and glutamate transporter activities, and mitochondrial function.

Direct comparisons of synaptic functional parameters in brain tissues from different groups of experimental animals and different samples from post mortem human brain are often hindered by the inability to perform assays at the same time. To circumvent these difficulties we developed methods for cryopreservation and long-term storage of neocortical synaptosomes. The synaptosomes are suspended in a cryopreservation medium containing 10% dimethylsulfoxide and 10% fetal bovine serum, and are slowly cooled to -80 degreesC and then stored in liquid nitrogen. The function of plasma membrane glucose and glutamate transporters, and mitochondrial electron transport activity and membrane potential were measured in fresh, cryopreserved (CP), and non-cryopreserved freeze-thawed (NC) synaptosomes. Glucose and glutamate transporter activities, and mitochondrial functional parameters in CP synaptosomes were essentially identical to those in fresh unfrozen synaptosomes. Glucose and glutamate transport were severely compromised in NC synaptosomes, whereas mitochondrial function and cellular esterase activity were largely maintained. Electron paramagnetic resonance studies in conjunction with a protein-specific spin label indicated that cryopreservation did not alter the physical state of synaptosomal membrane proteins. These methods provide the opportunity to generate stocks of functional synaptosomes from different experiments or post mortem samples collected over large time intervals.

ATP-Binding Cassette Transporters↗

PubMed1999

Effect of 2,6-diisopropylphenol and halogenated anesthetics on tetraphenylphosphonium uptake by rat brain synaptosomes: determination of membrane potential.

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP+) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202+/-8 mV (mean +/- SD) while the synaptosomal membrane showed a potential of 78+/-8 mV (mean +/- SD). When anesthetic concentrations (0.6-1 mM propofol, 10-40 microM enflurane, 30-50 microM isoflurane, 8-15 microM halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP+ taken up was observed as the anesthetic concentration was increased.

Anesthetics, Inhalation↗

PubMed1994

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus.

A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

Animals↗

PubMed1997

Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes.

Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 microM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4alpha-phorbol 12,13-didecanoate at 10 microM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity.

Animals↗

PubMed1983

An electron-spin-resonance spin-label study of the interaction of purified Mojave toxin with synaptosomal membranes from rat brain.

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.

Animals↗

PubMed1983

Dopamine uptake by rat striatal synaptosomes: time- and temperature-dependent decay and protection by dithiothreitol and dopamine.

The uptake of [3H]dopamine (DA) into rat striatal synaptosomes in the presence of a monoamine oxidase inhibitor was studied using a filtration technique. After a 10-min preincubation period, a fast initial uptake of [3H]DA was seen. Uptake reached a maximum after 4 min of incubation. If incubation was continued for more than 7 min, a gradual decrease in synaptosomal [3H]DA levels was found. Uptake was dependent on preincubation time; initial uptake velocity and maximal uptake decreased irreversibly with increasing preincubation periods. Moreover, the capacity of the synaptosomes to retain the [3H]DA during longer incubation times was progressively affected. The decrease in initial uptake activity was due to a decrease in the Vmax of the transport system. Dithiothreitol (2.8 mM) protected synaptosomal uptake activity against deterioration at 37 degrees C. Also, DA itself (10(-7)M) stabilized the uptake mechanism if added to the suspension before preincubation was started. Since [3H]DA uptake observed after loading the synaptosomes with labeled DA was similar to the uptake seen if the synaptosomes were not previously loaded with DA, it was concluded that under these conditions synaptosomal DA is completely exchangeable with exogenous substrate. Prolonged storage of the synaptosomes at 0 degree C also resulted in a time-dependent decrease in uptake activity (t1/2 = 116 min). The addition of unlabeled DA or dithiothreitol to the suspension did not affect instability at 0 degree C.

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