Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

Choline acetyltransferase binding to and release from membranes

1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.     

Binding of Clostridium botulinum neurotoxin to the presynaptic membrane in the central nervous system

Large synaptosome fractions were isolated from the cerebellar and cerebral cortices of rats and were incubated with Clostridium botulinum type A neurotoxin in vitro. The binding of the neurotoxin to the synapses was observed by electron microscopy, using the double-sandwich immunocytochemical method. Botulinum neurotoxin was preferentially bound to the presynaptic membrane in the large synaptosome fraction. The binding regions for the neurotoxin were localized on both the extrajunctional and junctional areas of the presynaptic membranes and appeared as patches of various sizes. However, they did not exist on the postsynaptic membranes. Botulinum neurotoxin is proposed to be a useful analytical tool for understanding the characteristics of the presynaptic membranes in the central nervous system.     

Acetylcholine in the brain of rats exposed to acute irradiation

A two-fold increase in acetylcholine, that can randomly be released by brain synaptosomes, is registered 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg; depolarization of the synaptosome membranes by potassium chloride increases the release of acetylcholine the augmentation of the release in this case being lower than that in the control. The initial rate of spontaneous neuromediator release from synaptosomes grows by 80 per cent whereas after depolarization of synaptosome membranes by potassium chloride, by 15 per cent. There is a 2.5-fold increase in the maximum rate of a highly specific uptake of choline with Km value being constant. Acetylcholine content of gray substance of irradiated rat brain is invariable.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Pyrene derivatives as markers of transbilayer effect of lipid peroxidation on neuronal membranes

Two different pyrene derivatives, namely 12-(1-pyrene)dodecanoic acid (P12-FA) and N-(12-(1-pyrene)dodecanoyl)-galactosylsphingosine I3-sulfate (P12-CS) have been used to follow lipid peroxidation both in model and natural membranes. The malondialdehyde (MDA) production in small unilamellar vesicles of dipalmitoylphosphatidylcholine/arachidonic acid (80:20, molar ratio), symmetrically labelled with both probes determined a progressive decrease of pyrene fluorescence due to an involvement of pyrene in the peroxidative reaction. Nervous membranes are particularly sensitive to lipid oxidation which differentially acts on the two layers of the membrane determining a greater rigidity of the exofacial one. Thus, we consider the possibility to asymmetrically introduce the pyrene ring, as P12-FA or P12-CS, in synaptosomes for monitoring lipid peroxidation in each layer of the membrane. The amount of the two probes incorporated in the membrane was 20 +/- 3 and 10 +/- 2 nmol/mg of protein for P12-FA and P12-CS, respectively. P12-FA was symmetrically distributed in the two layers, whereas 95% of P12-CS was incorporated in the exofacial layer of the membrane as determined by TNBS measurements. The decrease in fluorescence of synaptosome associated pyrene was, in the early stages of lipid peroxidation, greater for P12-CS than for P12-FA labelled membranes, indicating a greater susceptibility of the exofacial layer to iron-induced peroxidation.     

Effects of phospholipase A2 and alpha-tocopherol on the activity of adenylate cyclase of rat brain synaptosomes

The action of phospholipase A2 and alpha-tocopherol on adenylate cyclase system functioning and on the lipid bilayer microviscosity of the rat brain synaptosome membranes was investigated. It was shown that the exposure of the synaptosomes to phospholipase A2 increases the adenylate cyclase activity stimulated by guanylyl imidotriphosphate (GITP), decreases the adenylate cyclase activity stimulated both by isoproterenol and by isoproterenol with GITP. The preincubation of synaptosomes in medium containing alpha-tocopherol does not change the character of the phospholipase action on the adenylate cyclase activity stimulated by isoproterenol but normalizes the adenylate cyclase activity stimulated both by GITP and by GITP with isoproterenol. In the last case the normalizing action of alpha-tocopherol is not caused by alteration of the microviscosity of the lipid bilayer. It appears to be due to the modification of the lipid-protein interactions of annular lipids with activated complex of catalytic subunit and guanyl nucleotide-binding protein.     

Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid

The effects and modes of action of certain lipid second messengers and protein kinase C regulators, such as sphingosine, lysophosphatidylcholine (lyso-PC), and oleic acid, on Na,K-ATPase and sodium pump were examined. Inhibition of purified rat brain synaptosome Na,K-ATPase by these lipid metabolites, unlike that by ouabain, was subject to membrane dilution (i.e. inhibition being counteracted by increasing amounts of membrane lipids). Kinetic analysis, using the purified enzyme, indicated that sphingosine and lyso-PC were likely to interact, directly or indirectly, with Na+-binding sites of Na,K-ATPase located at the intracellular face of plasma membranes, a conclusion also supported by studies on Na,K-ATPase and 22Na uptake using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not sphingosine and lyso-PC) increased the affinity constant (K0.5) for K+, whereas sphingosine and lyso-PC (but not ouabain) increased K0.5 for Na+. Sphingosine and lyso-PC inhibited 86Rb uptake by intact human leukemia HL-60 cells at potencies comparable to those for inhibitions of purified Na,K-ATPase and protein kinase C. It is suggested that Na,K-ATPase (sodium pump) might represent an additional target system, besides protein kinase C, for sphingosine and possibly other lipid second messengers.     

Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy     

Determination of electrical characteristics of synaptosomal membranes of the brain

A correct method for evaluating the potential differences across plasma membranes of synaptosomes from the brain is presented. It takes into consideration the multicompartment synaptosome organization and is based on the accumulation of the radioactive permeant cation [3H]tetraphenylphosphonium. It is shown that upon potassium depolarization of the synaptosomes to about -5 mv there is a sharp decrease in the ion selectivity of the synaptic membranes.     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

Fatty acid release in incubations of serum with synaptosome and myelin subfractions of brain

To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in vitro at 37 degrees C, 0.43 and 0.26 mumol of fatty acid were released in incubations containing synaptosomes (1.37 mumols phospholipid) or myelin (1.23 mumols phospholipid), respectively, in the presence of 0.25 mL serum. Less than 0.05 mumol of fatty acid was liberated in incubations containing only serum, synaptosomes, or myelin. For synaptosomes and serum, docosahexaenoate was the principal fatty acid released (28 mol% of total) after 3 h of incubation. This fatty acid and arachidonate made up 43 mol% of the liberated fatty acid. The presence of free docosahexaenoate was of interest, as this fatty acid is particularly enriched in phosphatidylserine and phosphatidylethanolamine, phospholipids found in the cytoplasmic half of the synaptosomal plasma membrane and in interior synaptosomal membranes. In incubations of serum and myelin, oleate was the major free fatty acid produced in 30 min to 3 h of incubation (29-35 mol% of total). After 3 h, docosahexaenoate contributed 20 mol% to the total. The release of fatty acids from the membranes may be mediated by serum phospholipase(s) or possibly by activated endogenous lipolytic activities.     

Transbilayer distribution of alpha-tocopherol and lipid asymmetry in nerve tissue membranes

The alpha-tocopherol content and fatty acid composition of lipids in various types of nervous tissue membranes were studied. The transbilayer distribution of alpha-tocopherol and polyunsaturated fatty acids in liposomes and plasma membranes of synaptosomes was examined. It was shown that both phosphatidylethanolamine and phosphatidylserine are localized predominantly in the inner monolayer and they contain the bulk of polyenoic fatty acid residues. alpha-Tocopherol incorporated into liposomes from synaptosome plasma membrane lipids and present in synaptosome plasma membranes is also predominantly localized in the inner monolayers. No asymmetrical distribution of incorporated alpha-tocopherol was observed in liposomes prepared from a single phospholipid, e.g., dioleoylphosphatidylcholine.     

METABOLISM OF THE PHOSPHOINOSITIDES IN GUINEA-PIG BRAIN SYNAPTOSOMES

Abstract— The subcellular distribution of a number of enzymes concerned with inositol lipid metabolism has been studied in sub-fractions of disrupted guinea-pig brain synaptosomes. The enzymes were CDP-diglyceride: inositol phosphatidate transferase, phospha-tidylinositol kinase, diphosphoinositide kinase, diphosphoinositide phosphomonoesterase and diphosphoinositide diesterase. The distribution of phosphatidylinositol kinase in sub-fractions from water-treated synaptosomes was compared with that of other plasma membrane enzymes. After partial solubilization of synaptosomes by Triton X-100 the activities of phosphatidylinositol kinase and several other enzymes were examined.
Distribution of phosphatidylinositol kinase closely resembled that of acetylcholinesterase in sub-fractions of synaptosomes. Both enzymes appeared to be localised in the outer membrane of the synaptosome. CDP-diglyceride: inositol phosphatidate transferase was present in all types of synaptosomal membrane. All three enzymes concerned with diphosphoinositide metabolism were found in the cytoplasm of the synaptosome.     

The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes.

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.     

Initiation by estradiol of phosphoinositide catabolism in synaptosome membranes of the rat brain

A possibility of phosphatidylinositides catabolism initiation was revealed in the rat brain synaptosome membranes during in vitro action of estradiol. It was shown that appreciable enhancement of diacylglycerol yield was observed at already 5th second of initiation, which decreased then until the 30th second and kept the sufficiently high level. The yield of free arachidonic acid had also enhanced. Analogous dynamics of alteration was observed during the action of pharmacological as well as physiological concentrations of hormone.     

ATP-dependent Ca2+-transporting system in the rat brain during an early period of acute radiation injury

The rate of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation in a preparation of plasma membranes from brain synaptosomes increases 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg, a calcium sensitivity of both processes being increased. A unidirectional change in their kinetics indicates the early radiosensitivity of Ca2+ transfer systems in the brain synaptosome membranes. There is an increase in the availability of SH-groups of membrane preparation proteins for SH-reagents and in the sensitivity of Mg2+, Ca2+-ATPase reaction and ATP-dependent Ca2+ accumulation to trifluoperazine, a calmodulin inhibitor. Both processes lose their ability to be activated by exogenous calmodulin. It is suggested that at an early stage of radiation affection, a change occurs in the molecular organization of the ATPase-calmodulin membrane complex in plasma membranes of rat brain synaptosomes.     

Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin-Enriched Fractions of Bovine and Rat Brain

The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well-defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.     

Changes in the calcium metabolism of cerebral cortical structures in anoxia in vitro

Changes in membrane-bound calcium (Ca2+(b)) content in the brain cortex membrane structures were studied on subcellular fractions (synaptosomes, microsomes, mitochondria) during in vitro anoxia. The changes in Ca2+ content in hydrophobic domains of intracellular membranes were assessed, using chlorotetracycline fluorescent probe. It has been found that membranes of different neuronal compartments are not equally vulnerable to anoxia. A decrease in Ca2+9(b) content in response to anoxia occurs in synaptosomes and microsomes much sooner than in mitochondria. Therefore, Ca2+ release from intracellular membrane compartments, preceding the massive inward flow of extracellular Ca2+, seems to be one of those mechanisms initiating a complex range of intracellular reactions to disturbed oxygen supply in brain cortex neurons.     

Synaptic plasma membrane-bound acetylcholinesterase activity is not affected by docosahexaenoic acid-induced decrease in membrane order

We investigated the effect of administration of docosahexaenoic acid (C22:6, n-3; 300 mg/kg.day, for 12 weeks) on the degree of membrane order and membrane-bound acetylcholinesterase activity of the cerebral cortex synaptic plasma membrane in male Wistar rats. Docosahexaenoic acid levels in the synaptic plasma membrane increased significantly by 16% over levels in control rats concomitant with an increase in the molar ratio of docosahexaenoic acid to arachidonic acid. Synaptic plasma membrane order, assessed by 1,6-diphenyl-1,3,5-hexatriene, which measures order of the bulk internal hydrophobic lipid core, decreased significantly in the docosahexaenoic acid-fed rats. Lateral mobility of both global and annular lipids measured by pyrene also increased. Acetylcholinesterase activity of the synaptic plasma membrane was unaffected, and synaptic plasma membrane phospholipid contents increased in the docosahexaenoic acid-fed rats, with a concomitant decrease in the cholesterol/phospholipid molar ratio. Lipid peroxide and reactive oxygen species, indicators of tissue oxidative stress, decreased in both the cerebral cortex synaptosome and homogenate of the docosahexaenoic acid-fed rats. Arrhenius plot showed a break point in acetylcholinesterase activity at 22 degrees C and 24 degrees C in plasma membranes from docosahexaenoic acid-fed and control rats, respectively. The present experiment indicates that chronic administration of docosahexaenoic acid does not affect synaptic acetylcholinesterase activity and evoke oxidative stress, although it increases the disorder of the global and annular lipids of rat synaptic plasma membranes.