Synaptosomal Subfractions Research Articles

Evaluation of the Activity of Choline Acetyltransferase From Different Synaptosomal Fractions at the Distinct Stages of Spatial Learning in the Morris Water Maze.

Accumulated data have evidenced that brain cholinergic circuits play a crucial role in learning and memory; however, our knowledge about the participation of neocortical and hippocampal cholinergic systems in spatial learning needs to be refined. The aim of this study was to evaluate the association of the activity of membrane-bound and soluble choline acetyltransferase (ChAT) in the synaptosomal sub-fractions of the neocortex and hippocampus with performance of the spatial navigation task in the Morris water maze at different temporal stages of memory trace formation. To identify distinct stages of memory formation, rats were trained using a 5-day protocol with four trials per day. The mean escape latency for each trial was collected, and the entire dataset was subjected to principal component analysis. Based on the Morris water maze protocol, there were three relatively distinct stages of memory formation: days 1–2, day 3, and days 4–5. The remotely stored memory trace tested in repeated and reversal learning beginning on day 19 (14 days after the end of initial learning) was associated at the individual level mainly with performance during the second trial on day 21 (the third day or repeated or reversal learning). The ChAT activity data suggest the participation of cortical cholinergic projections mainly in the first stage of spatial learning (automatic sensory processing) and the involvement of hippocampal interneurons in the second stage (error-corrected learning). Cholinergic cortical interneurons participated mainly in the stage of asymptotic performance (days 4–5). It is advisable to evaluate other signalling pathways at the identified stages of memory formation.

Effects of succinate on ATP-induced Ca2+ waves in the nucleus accumbens

Event Abstract Back to Event Effects of succinate on ATP-induced Ca2+ waves in the nucleus accumbens Tünde Molnár1*, Peter Barabas1, Julianna Kardos1 and Miklos Palkovits2 1 Department of Neurochemistry, Institute of Biomolecular Chemistry, Hungarian Academy of Sciences, Hungary 2 Semmelweis University and Hungarian Academy of Sciences, Hungary We have previously reported a membrane protein that binds the intermediary metabolite of gamma-hydroxybutyric acid (GHB), succinic acid (SUC) and the gap-junction blocker carbenoxolone in nucleus accumbens (NA) synaptosomal subfractions. In the present study, we explored propagating Ca2+ waves induced by extracellular ATP (100 μM) and challenged them by possible inhibitors, intermediary metabolites and their combinations. Confocal laser imaging has been used to detect changes in the number and the fluorescence intensity of cells loaded with the fluorescent indicator of cytosolic Ca2+ ion Fluo-4 acetoxymethyl ester. We report for the NA, that ATP-induced Ca2+ wave propagates in astrocytes that can be decreased by gap junction blockers carbenoxolone (0.1 and 1 mM) and flufenamic acid (1 mM). Conversely the ATP-gated cation channel (P2x) antagonist suramin (0.1 mM) had no effect on the cell number and fluorescence intensity of cells of ATP-induced Ca2+ waves in the NA, suggesting that propagation depends on functional activity of gap-junctions. In addition, concentration-dependent effects of SUC and GHB were described. In low concentration (0.05 mM) SUC and GHB enhanced both the number and the fluorescence intensity of cells, while in higher concentration (0.2 and 2 mM) an inhibitory effect of SUC was observed. Our findings indicate that in the NA, calcium waves propagate via the mechanism involving gap-junctional connections between glutamate receptor containing astrocytes. The facilitatory role in astrocytic Ca2+ wave propagation suggests SUC as a strong candidate for metabolic signalling and will therefore benefit from further study. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Molnár T, Barabas P, Kardos J and Palkovits M (2009). Effects of succinate on ATP-induced Ca2+ waves in the nucleus accumbens. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.246 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2009; Published Online: 13 Mar 2009. * Correspondence: Tünde Molnár, Department of Neurochemistry, Institute of Biomolecular Chemistry, Hungarian Academy of Sciences, Budapest, Hungary, tmolnar@chemres.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tünde Molnár Peter Barabas Julianna Kardos Miklos Palkovits Google Tünde Molnár Peter Barabas Julianna Kardos Miklos Palkovits Google Scholar Tünde Molnár Peter Barabas Julianna Kardos Miklos Palkovits PubMed Tünde Molnár Peter Barabas Julianna Kardos Miklos Palkovits Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Emx2 homeodomain transcription factor interacts with eukaryotic translation initiation factor 4E (eIF4E) in the axons of olfactory sensory neurons.

We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.

Ryanodine receptor modulation by diadenosine polyphosphates in synaptosomal and microsomal preparations of rat brain

Diadenosine polyphosphates (Ap n As) are transmitter-like substances that act intracellularly via unclear mechanisms. Here we tested hypotheses that diadenosine tetraphosphate (Ap 4A) modulates ryanodine binding in microsomal and synaptosomal fractions of rat brain, and that Ap 4A affects modulation of ryanodine binding by divalent cations and caffeine. Using [ 3H]ryanodine-binding assays, we showed that Ap 4A produced significant and concentration-dependent increases in [ 3H]ryanodine binding in microsomes and these actions were reduced by Mg 2+ and potentiated by caffeine. In synaptosomal subfractions, effects of Ap 4A on [ 3H]ryanodine binding were most profound in subfractions enriched in synaptic vesicle-associated protein synaptophysin. These results suggest that Ap n As and ryanodine receptors are well placed to modulate Ca 2+-dependent synaptic processes.

Effect of reboxetine treatment on brain cAMP- and calcium/calmodulin-dependent protein kinases

Previous studies showed that the type II Ca 2+/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA) are affected by long-term antidepressant treatment in presynaptic and somatodendritic compartments, respectively. This study describes the long-term effects of the selective noradrenaline reuptake inhibitor reboxetine on PKA and CaMKII, in both the microtubule and subsynaptosomal fractions of rat brain. Unlike other antidepressants, chronic reboxetine induced in the cerebrocortical soluble and microtubule fractions a decrease in the [ 32P]cAMP binding to the type II PKA regulatory subunit. No change in the cAMP-dependent endogenous phosphorylation of the protein substrate, microtubule-associated protein 2 was observed. In the hippocampal subsynaptosomal fractions (synaptic vesicles and synaptosomal membranes) reboxetine induced a robust increase in the activity but not in the expression of CaMKII. An increase in the calcium/calmodulin-dependent phosphorylation of presynaptic substrates was also detected. These findings showed that reboxetine modulates post-receptor signal transduction systems in rat brain.

Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [14C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.

Subcellular localization of ryanodine receptors in rat brain

Subcellular fractions of rat brain were used to determine the subcellular localization of ryanodine receptors. [ 3H]Ryanodine binding in purified cortical, cerebellar and hippocampal synaptosomes was up to 3.6-fold higher compared with mitochondrial fractions. The density of sites ( B max) in hippocampal mossy fiber synaptosomes (249 fmol/mg protein) was 3.7-fold greater than in cortical synaptosomes (67 fmol/mg protein) and binding affinity was approximately 2-fold greater in the former ( K D, 6.1 nM) than the latter ( K D, 3.1 nM). At single sub-saturating concentrations of [ 3H]ryanodine, binding was 1.6-fold higher in mossy fibers compared with total hippocampal synaptosomes. [ 3H]Ryanodine binding sites were distributed similarly in subfractions of cortical synaptosomes and microsomes from discontinuous sucrose density gradients. An enrichment of sites was found in the lightest fractions containing the lowest activities of plasma membrane (5′-nucleotidase) and endoplasmic reticulum (glucose 6-phosphatase) enzyme markers when data for microsomal and synaptosomal subfractions were expressed as activity/binding per mg protein and when data for synaptosomal subfractions were expressed as a percentage of total activity/binding in collected fractions. Thus, ryanodine receptors appear to be concentrated in presynaptic terminals where they may play a major role in neurotransmitter release, and appear to be localized either in a specialized endoplasmic reticulum subcompartment or a distinct subcellular organelle.

Persistent Translocation of Ca2+/Calmodulin‐Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

The influence of brain ischemia on the subcellular distribution and activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca2+/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.

Synaptosomal non-mitochondrial ATPase activities: Age-related alterations by chronic normobaric intermittent hypoxia

In synaptosomes and synaptosomal subfractions (namely, synaptosomal plasma membranes and synaptic vesicles) the age-related alteration in the plasticity of synaptic energy-requiring ATPases (Na +, K +-ATPase, low- and high-affinity Ca 2+-ATPase, Mg 2+-ATPase and Ca 2+, Mg 2+-ATPase) were assayed in the cerebral cortex from 3- and 24-month-old normoxic rats and rats subjected to either mild or severe chronic (4 weeks) intermittent normobaric hypoxia. With the exception of the high-affinity Ca 2+-ATPase, aging induced a decrease in the ATPase activities from normoxic rats. The adaptation to mild hypoxia was characterized by an increase in the activity of Mg 2+-ATPase in 3-month-old rats, concomitant with a decrease in the activities of: (i) Na +,K +-ATPase and high-affinity Ca 2+-ATPase in both 3- and 24-month-old rats; and (ii) Ca 2+,Mg 2+-ATPase in 3-month-old ones. The adaptation to chronic intermittent severe hypoxia was characterized by a decrease in the activities of: (i) Na +,K +-ATPase, Ca 2+,Mg 2+-ATPase and high-affinity Ca 2+-ATPase in both 3- and 24-month-old rats and (ii) low-affinity Ca 2+-ATPase only in 24-month-old ones. The effect on Mg 2+-ATPase activity was characterized by a decrease in the activity of the enzymatic form located in the synaptic plasma membranes (involved in ATP hydrolysis to adenosine production), concomitant with an increase in the activity of the form located in the synaptic vesicles (involved in the turnover of transmitters, e.g., glutamate).

Age-related changes by hypoxia and trh analogue on synaptic atpase activities

Some synaptosomal energy-requiring ATPases were evaluated in the cerebral cortex from 3- and 24-month-old normoxic rats and rats submitted to either mild or severe chronic (4 weeks) intermittent normobaric hypoxia. Furthermore, 4-week treatment with saline or TRH analogue posatireline was performed. The activities of Na +,K +-ATPase, low- and high-affinity Ca 2+-ATPase, Mg 2+-ATPase, and Ca 2+,Mg 2+-ATPase were assayed in synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. With the exception of the high-affinity Ca 2+-ATPase, aging induced a decrea se in the ATPase activities from normoxic rats. The adaptation to chronic intermittent mild hypoxia was characterized by an increase in the activity of Mg 2+-ATPase in 3-month-old oats, concomitant with a decrease in the activities of: a) Na +,K +-ATPase and high-affinity Ca 2+-ATPase in both 3- and 24-month-old rats, and b) Ca 2+,Mg 2+-ATPase in 3-month-old ones. The TRH analogue posatireline increased the high-affinity Ca 2+-ATPase in both 3- and 24-month-old hypoxic rats, concomitant with an increase in Mg 2+-ATPase activity in 24-month-old ones. The adaptation to chronic intermittent severe hypoxia was characterized by a decrease in the activities of: a) Na +,K +-ATPase, Ca 2+,Mg 2+-ATPase and high-affinity Ca 2+-ATPase in both 3- and 24-month-old rats, and b) low-affinity Ca 2+-ATPase only in 24-month-old ones. The effect on Mg 2+-ATPase activity was characterized by a decrease in the enzymatic form located in the synaptic plasma membranes, concomitant with an increase in the form located in the synaptic vesicles. In both 3- and 24-month-old hypoxic rats, TRH analogue increased the activity of the Na +,K +-ATPase and the enzymatic form of the Mg 2+-ATPase located in the synaptosomal plasma membranes, concomitant with a decrease in the form of the Mg 2+-ATPase located in the synaptic vesicles.

Subcellular localization of the F5 protein to the neuronal membrane‐associated cytoskeleton

F5 was identified originally as an interleukin-2-regulated gene in the murine helper T-lymphocyte clone L2. Subsequent studies demonstrated high levels of F5 mRNA and protein in mature neurons in adult mouse central and peripheral nervous systems. The F5 protein was present in dendrites and perikarya but not in axons. In the present studies, the intracellular localization of the F5 protein in adult mouse brain was determined by subcellular fractionation and Western blotting. Although the deduced F5 sequence predicts a soluble protein, virtually no F5 immunoreactivity was found in the cytosol. The F5 protein was restricted to the P2 crude mitochondrial and P3 crude microsomal particulate fractions. Within the P2 fraction, F5 protein was enriched in the P2B synaptosomal subfraction. The results of temperature-dependent phase separation with Triton X-114 and alkaline extraction with sodium carbonate of the P2 and P3 fractions were consistent with the F5 protein being an extrinsic membrane-associated protein. Although essentially all of the F5 protein in the P3 fraction was membrane-associated, a substantial proportion of P2-associated F5 protein and nearly all of the synaptosomal F5 protein was detergent-insoluble. Direct isolation and subfractionation of brain cytoskeleton confirmed colocalization of F5 immunoreactivity with the membrane-associated cytoskeleton and postsynaptic densities. These studies suggest that the F5 protein, which has a large number of potential phosphorylation sites, plays a role in membrane-cytoskeletal interactions and in dynamic aspects of synaptic structure or function.

The subcellular distribution of [3H]-CGS 21680 binding sites in the rat striatum: Copurification with cholinergic nerve terminals

The subcellular distribution of the adenosine A2a receptor in rat striatum has been investigated using specific binding of the A2a-selective ligand [3H]-CGS 21680. After subcellular fractionation, the distribution of [3H]-CGS 21680 binding was similar to that of the cholinergic nerve terminal marker acetylcholinesterase rather than the more general membrane marker 5'-nucleotidase, with 42% of binding associated with the synaptosomal sub-fraction and 19% with a light membrane fraction. Binding of [3H]-CGS 21680 was also found to co-purify with the cholinergic nerve terminal marker choline acetyltransferase during immunoaffinity purification of striatal cholinergic nerve terminals. These results demonstrate that some adenosine A2a receptors are present on cholinergic nerve terminals in rat striatum.

Synaptosomal non-mitochondrial ATPase activities and drug treatment.

Energy-using non-mitochondrial ATPases were assayed in rat cerebral cortex synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The following enzyme activities were evaluated: Na+, K+ -ATPase; high- and low-affinity Ca2+ -ATPase; basal Mg(2+)-ATPase; Ca2+, Mg(2+)-ATPase. The evaluations were performed after four week-treatment with saline [controls] or alpha-adrenergic agents (delta-yohimbine, clonidine), energy-metabolism interfering compound (theniloxazine), and oxygen-partial pressure increasing agent (almitrine), in order to define the plasticity and the selective changes in individual ATPases. In rat cerebral cortex, the enzyme adaptation to four-week-treatment with delta-yohimbine or clonidine was characterized by increase in both high- and low-affinity Ca2+ -ATPase activities. The action involves the enzyme form located in the synaptic plasma membranes. The enzyme adaptation to the subchronic treatments with theniloxazine or almitrine was characterized by increase in Na+, K(+)-ATPase or Mg(2+)-ATPase activities, respectively. The action involves the enzymatic forms located in the synaptic plasma membranes. Thus, the pharmacodynamic effects of the agents tested should also be related to the changes induced in the activity of some specific synaptosomal non-mitochondrial ATPases.

Effect of intermittent mild hypoxia and drug treatment on synaptosomal nonmitochondrial ATPase activities

Synaptosomal nonmitochondrial ATPases linked to the energy-utilizing systems were evaluated in cerebral cortex from normoxic rats and rats submitted to mild intermittent normobaric hypoxia [12 hr daily exposure to N2:O2 (90:10) mixture for 4 weeks]. The activities of Na+,K(+)-ATPase; high- and low-affinity Ca(2+)-ATPase; basal Mg(2+)-ATPase; and Ca2+, Mg(2+)-ATPase were assayed in synaptosomes and synaptosomal subfractions, namely, synaptosomal plasma membranes and synaptic vesicles. The evaluations were performed either in normoxic rats or in hypoxic rats submitted to 4-week treatment with saline (controls) or a vasodilator agent (papaverine), an energy-metabolism interfering agent (theniloxazine), a calcium blocker (nicardipine), and a lipid-metabolism interfering agent (phosphatidylcholine) in order to define the plasticity and the selective changes in individual ATPases. In synaptosomes from rat cerebral cortex, the enzyme adaptation to the daily mild intermittent hypoxia for 4 weeks was characterized by an increase in the activity of Mg(2+)-ATPase, concomitant with a decrease in the activities of Na+,K(+)-ATPase, high-affinity Ca(2+)-ATPase, and Ca2+, Mg(2+)-ATPase. In hypoxic rats the enzyme adaptation to the 4-week treatment with phosphatidylcholine was characterized by an increase in Ca2+, Mg(2+)-ATPase activity and a decrease in Mg(2+)-ATPase activity. The action involves the enzymatic form located in the synaptic plasma membranes. In hypoxic rats the adaptation to the 4 week treatment with nicardipine was characterized by an increase in high-affinity Ca(2+)-ATPase activity, while the 4-week-treatment with theniloxazine induced an increase in Na+,K(+)-ATPase activity. The actions of both nicardipine and theniloxazine were related to the enzymatic forms located in the synaptic plasma membranes. The effects on the biophase induced by the sequential cycles of hypoxia/normoxia and the treatment with the various agents tested should also be related to the changes induced in the activity of some synaptosomal ATPases.

Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A2S on brain synaptic membranes

Crotoxin and other neurotoxic phospholipase A2s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several others to synaptic membranes has been demonstrated previously. In this study we used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After 125I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of approximately 60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein of approximately 45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of around 1 x 10(-8) M. Mojave toxin and some other phospholipase A2s were also highly inhibitory to this conjugation, and notexin and others were less effective, while beta-bungarotoxin and pancreatic PLA2 were totally ineffective. We concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A2s.

Prolonged ischemia differently affects phospholipase C acting against phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in brain subsynaptosomal fraction

The effect of 10 min ischemia on the activity of phospholipase C acting against [ 3H inositol-phosphatidylinositol (PI) and [ 3pH]inositol-phosphatidyl-inositol 4,5-bisphosphate (PIP 2) in the brain subsynaptosomal fractions was investigated. In the presence of endogenous CaCl 2, specific activity of phospholipase C acting on phosphatidylinositol was as follows: synaptic cytosol (SC) > synaptic vesicles (SV) > synaptic plasma membrane (SPM). Brain ischemia activated phospholipase C acting on PI by about 60% and 40% in SV and SPM, respectively. The enzyme of synaptic cytosol was not affected by ischemic insult. Phospholipase C acting against PIP 2 in the presence of endogenous calcium expressed the specific activity in the following order: SV > SPM > SC. After 10 min of brain ischemia, activity of phospholipase C acting on PIP 2 was significantly suppressed in all subsynaptosomal fractions by about 50–60%. These results indicate that prolonged ischemia produced activation exclusively of phospholipase C acting against phosphatidylinositol. Ischemia; Subsynaptosomal fraction; Phospholipase C

Synaptosomal "membrane-bound" choline acetyltransferase is most sensitive to inhibition by choline mustard.

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP‐Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.

Spermine binding to subsynaptosomal fractions of rat brain cortex

Binding sites for [14C]spermine have been identified in rat brain cortex subcellular fractions. The binding, characterized by using synaptosomal membranes, is specific for spermine. It was not detected below 20 degrees C and increased about three/four-fold with a temperature rise of 10 degrees C. Binding occurred only in the presence of -SH reducing agents. It was completely suppressed by metal chelating agents, and was stimulated about four-fold by 1-5 x 10(-5) M Fe2+. Smaller increases were observed in the presence of Mn2+, Ni2+, Ca2+, Mg2+, and Zn2+; in contrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn2+ = Ni2+ = Zn2+ = Co2+ much greater than Mg2+ greater than Ca2+). Bound radioactive spermine was not displaced by the addition of high concentrations of unlabelled polyamine or chelating agents, nor by precipitation and washing of the membranes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent beta-mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (less than 8,000) components, after SDS-polyacrylamide gel electrophoresis and fluorography. The Fe2+-stimulated [14C]spermine binding was neither influenced by a previous heating of the membranes at 100 degrees C for 30 min nor by trypsin or pronase digestion, whereas the heat-treatment increased the binding occurring in the absence of Fe2+ by about two fold. A non-enzymatic formation of a spermine-metal complex tightly bound to some membrane peptide(s) is suggested.

Subcellular site of action of imipramine in rodent brain.

To determine the site of action of imipramine, the subcellular distribution of [3H]imipramine in rodents was followed after both in vivo administration and in vitro incubation with tissue slices under "physiological" conditions. Total [3H]imipramine (10-1,000 nM) binding was associated with all primary fractions, but in particular with the nuclear (P1) and mitochondrial (P2) pellets and the synaptosomal (P2B) and myelin (P2A) fractions. Using an excess of imipramine to define any nonspecific interactions, a specific association was observed mainly in those fractions containing isolated nerve terminals and to a lesser extent with the purified myelin fraction. Preparation of subsynaptosomal fractions by osmotic lysis indicated that [3H]imipramine was associated with the synaptic vesicle and microsomal fractions and also with synaptosomal membranes. The degree of binding to the vesicular and microsomal fractions was increased with the length of preparation time, whereas there was an inverse relationship between the length of preparation and the amount bound to the synaptosomal membrane fraction. There was no evidence of an intrasynaptosomal accumulation of [3H]imipramine at concentrations up to 1,000 nM. [3H]2-Nitroimipramine, a slowly dissociating imipramine derivative, was exclusively located in synaptic membrane fractions. Prior treatment of rats with a combination of 5,7-dihydroxytryptamine and desipramine reduced 5-hydroxytryptamine levels and the levels of [3H]imipramine associated with the synaptosomal fractions to the same extent. It is concluded that imipramine is associated with a binding site localised on 5-hydroxytryptaminergic nerve terminals and that there is a redistribution to other sites (vesicular and microsomal) during the isolation procedure.