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PubMed1991

Effect of ethanol on calcium-uptake and phospholipid turnover by stimulation of adrenoceptors and muscarinic receptors in mouse brain and heart synaptosomes.

The effect of ethanol treatment on mouse brain and heart synaptosomal 45Ca uptake and the incorporation of [3H]inositol and [14C]choline into phosphatidylinositol (PI) and phosphatidylcholine (PC) were investigated. Ethanol in drinking water (15%) was given to mice for 3 weeks. The consumption of ethanol increased gradually during treatment but food intake was almost the same as control. The body weight of ethanol-treated mice was slightly less than that of control. The synaptosomal lipid peroxidation level of ethanol-treated mice was almost the same as control in the brain and heart. On the other hand, the synaptosomal glutathione level of ethanol-treated mice was higher than control in both brain and heart. The 45Ca uptake of brain and heart from ethanol-treated mice was 87% and 216% of control mice, respectively. Not only ethanol but also norepinephrine (NE), carbachol (Carb), or isoproterenol (IsoPro) added in vitro increased 45Ca uptake in all cases. The incorporation of [3H]inositol into PI in the brain and heart synaptosomes of ethanol-treated mice was 150% and 113% of control, respectively. The incorporation of [14C]choline into PC in the brain and heart of ethanol-treated mice was 104% and 125% of control, respectively. In vitro addition of ethanol, NE, Carb or IsoPro to brain synaptosomes increased the incorporation of [3H]inositol and [14C]choline into PI and PC, respectively, in both control and ethanol-treated mice. In the case of heart synaptosomes, NE and Carb increased the incorporation of [3H]inositol and [14C]choline into phospholipids in control mice but not ethanol-treated mice. However, IsoPro increased the incorporation by both control and ethanol-treated heart synaptosomes. These results suggest that alpha-adrenoceptors and the cholinergic system of the heart play important roles in modulating the toxic effects of ethanol.

Animals↗

PubMed1984

Proline, glutamate and glutamine metabolism in mouse brain synaptosomes.

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)

Amino Acids↗

PubMed1989

Tetrahydroaminoacridine but not 4-aminopyridine inhibits high-affinity choline uptake in striatal and hippocampal synaptosomes.

We have compared the effects of tetrahydroaminoacridine (THA), 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on high affinity choline uptake and the release of newly synthesized acetylcholine (ACh) from striatal and hippocampal synaptosomes, and on choline acetyltransferase (ChAT) activity in rat striatal synaptosomes. Incubation of the striatal synaptosomes with various THA concentrations (5-100 microM) caused a concentration-dependent inhibition in their accumulation of soluble 14C-labeled compounds ([14C]choline and [14C]ACh); at concentrations of 50 microM or greater the THA also completely suppressed the release of newly synthesized [14C]ACh. 4-AP slightly but significantly decreased the accumulation of the [14C]ACh in the striatal synaptosomes, without affecting that of [14C]choline, but markedly increased the release of [14C]ACh into the medium; hence the drug stimulated net choline uptake (by 19, 20 and 31%, respectively, in the presence of 5, 50 and 100 microM concentrations). Like THA, but not 4-AP, TEA decreased both the accumulation of soluble 14C-compounds in the striatal synaptosomes and the release of newly synthesized [14C]ACh. Similar effects of THA and 4-AP on HACU and the release of [14C]ACh were also observed when hippocampal synaptosomes were used. THA and 4-AP are structurally similar, and share with TEA the ability to block certain potassium channels. Present data show that, in spite of these similarities, these compounds produce opposite effects on net choline uptake by striatal and hippocampal synaptosomes.

4-Aminopyridine↗

PubMed1991

Opiate tolerance-induced modulation of free intracellular calcium in synaptosomes.

Synaptosomes were prepared from morphine-tolerant and non-tolerant mice. Significantly higher levels of basal free intracellular calcium were observed in the synaptosomes from the opiate-tolerant mice compared to synaptosomes from non-tolerant mice (468 nM versus 328 nM, respectively). In addition, morphine (1 microM) failed to attenuate KCl-induced rises in intracellular calcium in the synaptosomes from the tolerant mice. Conversely, morphine produced a concentration-related, naloxone-reversible attenuation of 50 mM KCl-induced rises in intracellular calcium in the synaptosomes from the non-tolerant mice. Omega conotoxin, which blocks both "L" and "N" type calcium channels, attenuated KCl-stimulated rises in intracellular calcium only in synaptosomes from non-tolerant mice. BAY-K 8644, an "L-type" calcium channel agonist, produced nifedipine-reversible increases in intracellular calcium in the synaptosomes from the tolerant animals only. These data suggest that chronic exposure to morphine results in an alteration in either the number of the activation state of calcium channels in the membrane. Changes in intracellular free calcium may be the final common pathway through which neurons adapt to the chronic exposure to morphine.

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

PubMed1987

Interactions of the neurotoxic complex from the venom of the false horned viper (Pseudocerastes fieldi) with rat striatal synaptosomes.

The very high lethal potency of the neurotoxic complex (Cb) from the venom of Pseudocerastes fieldi following direct administration into the lateral ventricle of the brain, as compared with potency following i.v. administration, suggests that the toxin acts on the central nervous system. Rat striatal synaptosomes were selected to study interactions of the toxin with nerve endings. CbII, the toxic phospholipase A2 component of the toxin, as well as the reconstituted complex (CbI + CbII), inhibited the high affinity choline transport into synaptosomes. Fifty per cent inhibition was obtained at 10 nM CbII after 20 min preincubation of the synaptosomes at 37 degrees C. Choline uptake was inhibited under conditions of minimal leakage of lactate dehydrogenase and probably very low phospholipase A2 activity (in the absence of Ca2+ with Sr2+ or with EGTA). The inhibition of choline uptake was irreversible and was evident after a short preincubation at 0 degrees C. CbII also enhanced the release of acetylcholine from synaptosomes preloaded with labelled choline, but this effect was markedly reduced in the presence of the acidic component (CbI) of the complex. Binding of 125I-CbII could be demonstrated with synaptosomes and with erythrocytes, however, the reconstituted complex (CbI + CbII) was bound only by the synaptosomes, though less effectively than free 125I-CbII. An increased specific binding was evident with purified synaptosomes as compared with a crude preparation. These results support the notion that the non-toxic subunit increases the specificity of the toxic phospholipase A2.

Animals↗

PubMed1996

Role of transglutaminase in [3H]5-HT release from synaptosomes and in the inhibitory effect of tetanus toxin.

It has been suggested that the Ca(2+)-dependent enzyme transglutaminase (TGase) may suppress vesicular neurotransmitter release and mediate the inhibitory effect of tetanus toxin (TetTx) on exocytosis. The aim of the present study was to test this in a model of [3H]5-HT release from superfused rat cortical synaptosomes. Monodansylcadaverine, a synthetic amine that acts as an alternative substrate for TGase, showed dose-dependent releasing activity which, however, was Ca(2+)-independent, being maintained in a Ca(2+)-free buffer (containing EGTA) or using synaptosomes preloaded with the intracellular Ca2+ chelator BAPTA. Preincubation of synaptosomes with RS-48373, an irreversible TGase inactivator, resulted in marked (64%) and persistent inhibition of endogenous TGase but did not alter basal and K(+)-induced [3H]5-HT release. Preincubation of synaptosomes with 10 nM TetTx resulted in 52% inhibition of K(+)-induced [3H]5-HT release, and this effect was not antagonized in RS-48373-treated synaptosomes. The inhibitory effect of TetTx was significantly antagonized by 20 mM captopril, a metalloendoprotease inhibitor, confirming in rat brain synaptosomes that TetTx inhibits exocytosis by acting as a metalloendoprotease. These results suggest that TGase is not involved in controlling [3H]5-HT release from resting and depolarized synaptosomes, or in the inhibitory effect of TetTx.

Animals↗

PubMed1987

Binding of specific glycoconjugates to human brain synaptosomes: studies using glycosylated beta-galactosidase.

Using glycosylated beta-galactosidase (beta-gal) as a glycoprotein model, binding of glycoconjugates to human brain synaptosomes was studied. Out of beta-gal modified with a series of p-aminophenyl alpha- or beta-glycosides, beta-gal modified with p-aminophenyl beta-D-glucopyranoside (beta-D-Glc beta-gal) was bound to the synaptosomes most effectively, then beta-gal modified with beta-D-galactoside and with alpha-D-mannoside. Kinetic studies on the binding of beta-D-Glc beta-gal indicated the presence of saturable binding on human brain synaptosomes. The values of the apparent Km and of the maximal binding velocity were obtained to be 248 +/- 32.9 microM and 43.8 +/- 1.43 fmol/min/mg synaptosome protein, at 4 degrees C and pH 7.5. The specificity of the sugar recognition site proved by the competitive inhibition of the binding of beta-D-Glc beta-gal by bovine serum albumin modified with the same glycoside. The binding of beta-gal modified with beta-D-galactoside was inhibited by treatment of the synaptosomes with trypsin, phospholipase A2, C and D, and with neuraminidase, while the binding of beta-D-Glc beta-gal was inhibited by neuraminidase treatment of synaptosomes. In subcellular fractions of human brain the binding protein was localized mainly in synaptosomes.

Cerebral Cortex↗

PubMed1994

Amino acids differentially inhibit the L-[3H]arginine transport and nitric oxide synthase in rat brain synaptosomes.

The nitric oxide synthase (NOS) present in the cytosol obtained from rat cerebral cortex synaptosomes was inhibited by NG-nitro-L-arginine (L-NOArg) with an IC50 value of approximately 0.06 microM. This compound did not affect the transport of L-arginine (L-Arg) into synaptosomes at concentrations up to 100 microM but other potential inhibitors of NOS (NG-monomethyl-L-arginine, NG-amino-L-arginine and L-arginine methyl ester) inhibited L-Arg transport at a concentration < 5 microM. We showed that concentrations of L-NOArg (0.001-3 microM) that did not block the uptake of tritiated arginine (L-[3H]Arg) inhibited the catalytic activity of NOS in intact synaptosomes. L-NOArg at a concentration of 1 microM inhibited the cytosolic enzyme by 98.0 +/- 2.0% of the total NOS activity whereas the enzyme studied in the intact synaptosomes was only inhibited by 75 +/- 5% which suggested that the NOS in synaptosomes is not fully accessible to the external L-NOArg. On the other hand, L-Lysine did not inhibit the cytosolic NOS activity of ruptured synaptosomes but at a concentration that blocked 50.0 +/- 4.5% of L-[3H]Arg uptake it inhibited the NOS activity in intact synaptosomes by 12.6 +/- 3.6%, suggesting that the transport of L-Arg may be an important regulatory step in the pathway for nitric oxide generation.

Amino Acid Oxidoreductases↗

PubMed1986

Uptake of serotonin into rat platelets and synaptosomes: comparative structure-activity relationships, energetics and evaluation of the effects of acute and chronic nortriptyline administration.

In order to establish whether the uptake systems in platelets and synaptosomes are equivalent, the structure-activity relationships for drug-induced inhibition of serotonin uptake were examined in vitro. The rank order for potency of inhibitors was the same in platelets as in synaptosomes, namely imipramine greater than nortriptyline greater than desmethylimipramine much much greater than norepinephrine greater than histamine; in addition, serotonin uptake was clearly distinguishable from the norepinephrine synaptic uptake mechanism, which displayed a different rank order. The synaptosomal uptake of serotonin was, however, much more dependent upon maintenance of Na+-K+-ATPase activity for its energy source than was the platelet uptake mechanism. Acute administration of nortriptyline produced substantial inhibition of platelet serotonin uptake and a smaller degree of inhibition of synaptosomal uptake; inhibition was detectable even after extensive washing of the platelet and synaptosome preparations, and was associated with persistent binding of the drug to the organelles. Chronic infusion of nortriptyline (20 mg/kg for 21 days, followed by a 24 hr washout period to dissipate persistent binding) did not alter the uptake capacity of synaptosomal or platelet preparations, but did cause a shift in the drug specificity of inhibitors. The latter effect was in opposite directions in platelets vs. synaptosomes. These data indicate that the platelet uptake mechanism does bear some resemblance to that seen in serotonergic neurons, but that the energy source for transport differs and the two mechanisms respond differently to prolonged drug administration in vivo. The use of uptake as a marker during the course of antidepressant administration is likely to be confounded by persistent direct drug effects on these organelles.

Animals↗

PubMed1980

Inhibition of mouse brain synaptosomal ATPases and ouabain binding by chlordecone.

The effect of chlordecone on the mouse brain synaptosomal Na+-K+ ATPase, Mg2+ ATPase and p-nitrophenyl phosphatase (PNPPase) activities was determined. In addition, the effect of chlordecone on [3H] ouabain binding to synaptosomes was also investigated. The in vitro data show that chlordecone inhibits PNPPase, Na+-K+ ATPase, and Mg2+ ATPase activities with ID50 values of 4, 5 and 7 micrograms respectively. Treatment of mice with symptomatogenic doses of chlordecone resulted in a decreased synaptosomal Na+-K+ and oligomycin-sensitive (mitochondrial) Mg2+ ATPases. The decrease was dose-dependent. The oligomycin-insensitive Mg2+ ATPase activity was unaffected either in vitro or in vivo. The binding of [3H] ouabain to synaptosomal membranes was inhibited by chlordecone in a dose-dependent manner in both in vitro and in vivo experiments. The binding of [14C] chlordecone to synaptosomes occurs even at nanomolar concentrations. The marked inhibition of brain synaptosomal ATPases and ouabain binding by chlordecone suggests that chlordecone may impair active transport mechanisms in synaptosomal membranes.

4-Nitrophenylphosphatase↗

PubMed1990

Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine.

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (-)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40-50% inactivation of Gi and Go proteins, which is evident from the reduction of ADP ribosylation with [32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.

Adenosine Diphosphate↗

PubMed2004

Mechanisms underlying the riluzole inhibition of glutamate release from rat cerebral cortex nerve terminals (synaptosomes).

We have examined the effect of riluzole, a neuroprotective agent with anticonvulsant properties, on the release of endogenous glutamate from rat cerebrocortical synaptosomes using an on-line enzyme-coupled fluorometric assay. Riluzole inhibited the calcium-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine, and this presynaptic inhibition was concentration-dependent. Riluzole did not alter either 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or ionomycin-mediated glutamate release, indicating that riluzole-mediated inhibition of glutamate release is not due to a decrease in synaptosomal excitability or a direct effect on the exocytotic machinery. Examination of the effect of riluzole on Ca2+ influx revealed that the diminution of glutamate release could be attributed to a reduction in cytosolic calcium. A possible effect of riluzole on synaptosomal calcium channels was confirmed in experiments where synaptosomes pretreated with P/Q-type calcium channel blocker omega-agatoxin IVA, which abolished the riluzole-mediated inhibition of glutamate release. In addition, pretreatment of synaptosomes with either the Gi/Go protein inhibitor pertussis toxin or the GABAB receptor agonist baclofen, completely prevented the inhibitory effect of riluzole on 4-aminopyridine-evoked glutamate release. It is concluded that riluzole exerts their presynaptic inhibition, likely through a reduction in the calcium influx mediated by P/Q-type calcium channels, and thereby inhibits the release of glutamate from rat cerebrocortical nerve terminals. This release inhibition may involve a pertussis toxin-sensitive G protein signalling pathway. This finding provides further support that presynaptic calcium channel blockade concomitant with inhibition of glutamate release could be an important mechanism underlying the therapeutic actions of this drug.

Animals↗

PubMed1998

[3H]-noradrenaline secretion from rat cortex synaptosomes perforated with Staphylococcus aureus alpha-toxin.

Rat cortex synaptosomes have been successfully perforated with high concentrations (> or = 400 U/ml) of Staphylococcus aureus alpha-toxin. The free Ca2+-concentration dependence of [3H]-noradrenaline release was similar to that observed for PC 12 and chromaffin cells. Release from the alpha-toxin perforated synaptosomes was not significantly inhibited by omega-conotoxin GVIA. Initially, Ca2+-dependent release was independent of MgATP (for 0.5 min), but became increasingly dependent on MgATP with time. Lactate dehydrogenase efflux from alpha-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein did not enter the synaptosomes. [3H]-noradrenaline release was temperature and alpha-toxin-concentration dependent. Ca2-dependent release was more resistant to rundown from alpha-toxin- than from streptolysin-O-perforated synaptosomes. This preparation of perforated synaptosomes should be useful for studies of regulated exocytosis from nerve endings.

Adenosine Triphosphate↗

PubMed2002

Aluminum accumulation and membrane fluidity alteration in synaptosomes isolated from rat brain cortex following aluminum ingestion: effect of cholesterol.

In the present work, we studied the effect of cholesterol/phospholipid (CH/PL) molar ratio on aluminum accumulation and aluminum-induced alteration of membrane fluidity in rat brain cortex synaptosomes. We observed that sub-acute (daily supply of 1.00 g of AlCl(3) during 10 days) and chronic (daily supply of 0.03 g of AlCl(3) during 4 months) exposure to dietary aluminum leads to a synaptosomal aluminum enrichment of 45 and 59%, respectively. During chronic exposure to AlCl(3), the enhancement of aluminum content was prevented by administration of colestipol (0.31 g/day), which decreased the synaptosomal membrane CH/PL molar ratio (nmol/nmol) from 1.2 to 0.4. Fluorescence anisotropy analysis, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), showed that after treatment with colestipol a decrease in membrane order occurs at the level of hydrophilic lipid-water surface and deeper hydrophobic region of the synaptosomal membrane. When the rats were exposed to aluminum, it was observed a significant enhancement of membrane fluidity, which was more pronounced at the level of the membrane hydrophilic regions. Meanwhile, when chronic exposure to dietary AlCl(3) was accompanied by treatment with colestipol, the aluminum-induced decrease in membrane order was negligible when compared to TMA-DPH and DPH anisotropy values measured upon colestipol treatment. In contrast, in vitro incubation of synaptosomes (isolated from control rats) with AlCl(3) induced a concentration-dependent rigidification of this more hydrophilic membrane region. The opposite action of aluminum on synaptosomal membrane fluidity, during in vivo and in vitro experiments, appears to be explained by alteration of synaptosomal CH/PL molar ratio, since a significant reduction (approximately 80%) of this parameter occurs during in vivo exposure to aluminum. In conclusion, during in vivo exposure to aluminum, fluidification of hydrophilic regions and reduction of CH/PL molar ratio of presynaptic membranes accompany the accumulation of this cation, which appear to restrict aluminum retention in brain cortex nerve terminals.

Aluminum↗

PubMed1997

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes.

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.

2-Chloroadenosine↗

PubMed1988

Endogenous glycosphingolipid acceptor specificity of sialosyltransferase systems in intact Golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain.

Preparations highly enriched in Golgi complex membranes, synaptosomes, and synaptic plasma membranes (SPM) by marker enzyme analysis and electron microscopic morphology were made from the brains of 28-day-old rats. These were incubated with cytidine 5'-monophosphate-N-acetyl[14C]neuraminic acid (CMP-NeuAc) in a physiologic buffer, without detergents. Glycolipid sialosyltransferase activities (SATs) were measured by analyzing incorporation of radiolabeled NeuAc into endogenous membrane gangliosides. Golgi SAT was diversified in producing all the various molecular species of labeled gangliosides [2.64 pmol of NeuAc transferred (mg of protein)-1 h-1]. Synaptosomal SAT exhibited a lower activity [0.66 pmol (mg of protein)-1 h-1], but it was highly specific in its labeling pattern, with a marked preference for labeling NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1 Cer (GD3 ganglioside). SPM prepared from the synaptosomes retained the GD3-related SAT (or SAT-2), and the total specific activity increased [1.41 pmol (mg of protein)-1 h-1], which suggests that the location of the synaptosomal activity is in the SPM. These results indicate that SAT activity in Golgi membranes differs from that in synaptosomes with regard to endogenous acceptor substrate specificity and SAT activity of synaptosomes should be located in the synaptosomal plasma membrane. This SAT could function as an ectoenzyme in concert with ecto-sialidase to modulate the GD3 and other ganglioside population in situ at the SPM of the central nervous system.

Brain↗

PubMed2002

Oxidative stress in synaptosomal proteins from mutant presenilin-1 knock-in mice: implications for familial Alzheimer's disease.

Presenilin-1 (PS-1) is a transmembrane protein that may be involved in the processing of amyloid precursor protein (APP). Mutations in PS-1 are the major cause of familial Alzheimer's disease (AD). AD brain is under significant oxidative stress, including protein oxidation. In the present study, protein oxidation was compared in synaptosomes from knock-in mice expressing mutant human PS-I (M146V mutation) and from wild-type mice expressing non-mutant human PS-1. Synaptosomal membrane protein conformational alterations associated with oxidative stress were measured using electron paramagnetic resonance (EPR) in conjunction with a protein-specific spin-label. Direct synaptosomal protein oxidation was assessed by a carbonyl detection assay. Synaptosomal proteins from PS-1 mutant mice displayed increased oxidative stress as measured by both techniques, compared with synaptosomal proteins from wild type mice. These data suggest that PS-1 mutations cause oxidative alterations in synaptosomal membrane protein structure and oxidative modification of synaptosomal proteins. Our findings suggest that familial AD may be associated with oxidative stress that may play a pivotal role in neuronal dysfunction and death.

Alzheimer Disease↗

PubMed1999

Energy metabolism of synaptosomal subpopulations from different neuronal systems of rat hippocampus: effect of L-acetylcarnitine administration in vivo.

The maximum rate (Vmax) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as "large" and "small" synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatment with L-acetylcarnitine at two different dose levels (30 and 60 mg x kg(-1), 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of "small" synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.

Acetylcarnitine↗