SEARCH · PubMed Health

Results for “synaptosomes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources

PubMed1997

Characterization of melittin effects in synaptosomes.

The effects of melittin at increasing concentrations on: [3H]GABA release from mouse brain synaptosomes; on the radioactivity released from [3H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 microM progressively increases [3H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [3H]GABA induced by melittin below 3 microM is Ca2+ dependent, but not that induced by the higher concentrations. The Ca2+ dependent fraction of the [3H]GABA released by 0.3 microM melittin is selectively inhibited by 10 microM quinacrine and 1 microM nordihydroguaiaretic acid (NDGA) and facilitated by 3 microM indomethacin, whereas the Ca2+ independent fraction of the [3H]GABA released by melittin is not. In the presence of Ca2+, melittin 0.3, 1 and 10 microM progressively increases [3H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 microM treated synaptosomes, but a noticeable disorganization is produced in 10 microM melittin-treated synaptosomes, independently on the presence of external Ca2+. LDH activity only increases over control activity in the supernatant solutions of 10 microM melittin treated synaptosomes, also in a Ca2+ independent manner. Our interpretation of these results is that the Ca2+-dependent, pharmacologic sensitive component of melittin-induced release of [3H]GABA, unmasked when 0.3 microM melittin was used, involves the activation of a Ca2+-dependent type of membrane PLA2. The Ca2+-independent release of [3H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.

Animals↗

PubMedDate not supplied

Co-release of glutamate and aspartate from cholinergic and GABAergic synaptosomes.

Glutamate and aspartate are known to be released in a calcium-dependent fashion by depolarizing stimulation of mammalian brain synaptosomes (isolated nerve endings), an observation which strengthens their claims to be neurotransmitter candidates. The source of these compounds has been interpreted as the exclusively glutamatergic or aspartatergic synaptosome sub-populations assumed to be present in the standard heterogeneous preparations from mammalian brain. Several neurotransmitter-specific synaptosomal surface markers have recently been identified by immunolysis studies and these have allowed separation of subpopulations of synaptosomes by an affinity purification method. These markers appear to be closely related to the biosynthetic enzyme for the principal neurotransmitter released by each sub-category of synaptosome. We have isolated highly purified, metabolically active, GABAergic and cholinergic synaptosomes from cerebral cortex using antisera recognizing either glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT), in conjunction with magnetic microspheres covalently coupled to Protein A (ref. 8), and now report that these synaptosomes release both glutamate and aspartate, in addition to their principal neurotransmitter, when treated with chemical depolarizing agents.

Acetylcholine↗

PubMed1997

Characterization of [35S]-ATP alpha S and [3H]-alpha, beta-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations.

1. We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATP alpha S and [3H]-alpha, beta-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. 2. Synaptosomes possess sites with high affinity for [35S]-ATP alpha S (Kd = 22.2 +/- 9.1 nM, Bmax = 14.8 pmol mg-1 protein). The rank order of the competition potency of the different compounds (ATP alpha S, ATP, ATP gamma S > ADP beta S, 2-MeSATP > deoxyATP, ADP > > UTP, alpha, beta-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. 3. Under identical conditions [35S]-ATP alpha S and [3H]-alpha, beta-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-alpha, beta-MeATP binding sites (Kd = 13.7 +/- 1.8 nM, Bmax = 6.34 +/- 0.28 pmol mg-1 protein) was 38 fold higher than the potency of alpha, beta-MeATP to displace [35S]-ATP alpha S binding (Ki = 0.52 microM). ATP and ADP beta S competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2-MeSATP, UTP, GTP) did not affect specific [35H]-alpha, beta-MeATP binding at concentrations up to 100 microM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. 4. Binding of [35S]-ATP alpha S on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+). 5. The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-alpha, beta-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10 microM to 1 mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [35S]-ATP alpha S binding was not influenced at the same range of Ca2+ or Mg2+ concentrations (10 microM to 1 mM). The addition of Mg2+ (5 mM) increased the affinity of [35S]-ATP alpha S for the high affinity site 10 fold. 6. Diadenosine polyphosphates had a bimodal effect on [35S]-ATP alpha S binding to synaptosomal membranes. AP5A and Ap6A enhanced binding of [35S]-ATP alpha S 1.6 fold in a concentration range between 0.1 and 50 microM. Ap3A was a weak inhibitor with a Ki value of 7.2 microM. Ap4A, AP5A and Ap6A inhibited with Ki values > 100 microM. These data support the concept that diadenosine polyphosphates do not directly interact with ATP alpha S binding sites. 7. In conclusion, on the basis of present knowledge of the interaction of P2-purinoceptor active compounds with P2x- and/or P2Y-purinoceptors, our data strongly suggest that [35S]-ATP alpha S is a useful tool to study P2Y-purinoceptors. Thus, the [35S]-ATP alpha S binding site might to a large extent represent P2Y-purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg2+ on binding.

Adenosine Triphosphate↗

PubMed2000

Cannabinoid receptor-mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes.

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically-evoked [(3)H]-acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes. [(3)H]-ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium-free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs. In hippocampal synaptosomes the 1.3 mM calcium-evoked release and the high potassium-evoked [(3)H]-ACh release were inhibited by the cannabinoid agonist, WIN 55212-2, by 59 and 39%, respectively, and with an EC(50) of approximately 1 nM. WIN 55212-2 produced a similar, although less potent, inhibition of [(3)H]-ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212-2 on [(3)H]-ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices. The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium-evoked [(3)H]-ACh release in hippocampal synaptosomes (EC(50)<0.3 nM) but had no effect on potassium-evoked release or on [(3)H]-ACh release in the cortex or striatum. In conclusion our data demonstrates the inhibitory effects of WIN 55212-2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A-induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.

Acetylcholine↗

PubMed1995

Pharmacological characterization of the voltage-dependent Ca2+ channels present in synaptosomes from rat and chicken central nervous system.

The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (omega-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and omega-agatoxin IVA]. K(+)-induced Ca2+ uptake by chicken synaptosomes was blocked by omega-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 microM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and omega-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 microliter/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and omega-agatoxin IVA affected Ca2+ uptake, FTX (3 microliters/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with omega-agatoxin IVA saturation was not reached even at 5 microM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 microliter/ml) and different concentrations of omega-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and omega-conotoxin GVIA was never greater than the one observed with omega-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by omega-conotoxin MVIID (1 microM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 microM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice.

Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta-peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.

Amyloid beta-Peptides↗

PubMed1988

Characterization of dihydropyridine-sensitive calcium channels in rat brain synaptosomes.

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.

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

PubMed1992

Effect of age on potassium- and tyramine-induced release of norepinephrine from cardiac synaptosomes in male F344 rats.

Potassium (K+)-induced norepinephrine (NE) release was examined in preparations of cardiac synaptosomes and sliced atria from 6-, 24-, and 26-mo-old male F344 rats. Cardiac synaptosomes were prepared from rat hearts by collagenase digestion followed by homogenization in 0.32 M sucrose and centrifugation. The synaptosome preparations and the sliced atria were labeled with 3H-NE and then placed in a superfusion system. K(+)-induced net fractional release of NE from synaptosomes prepared from 24- and 26-mo-old rats (4.3% and 3.0%, respectively) was significantly reduced when compared to NE release from synaptosomes from 6-mo-old rats (5.2%). K(+)-induced NE release from sliced atria from 24-mo-old rats (4.7%) was also significantly reduced when compared to NE release from atria from 6-mo-old rats (6.3%). Perfusion of cardiac synaptosomes with buffer prepared without calcium (CA++free, < 5 microM) reduced K(+)-induced release by 50% in all age groups studied. Perfusion with tyramine induced identical rates of NE release from cardiac synaptosomes prepared from 6- and 24-mo-old rats. These results confirm that depolarization-induced NE release from cardiac sympathetic nerves is reduced in the old male F344 rat.

Adrenergic Fibers↗

PubMed2000

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells.

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (rho degrees ) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA(Arg)genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA Mbo I polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient's brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.

Adult↗

PubMed1981

Newly synthesized L-[14C]dopamine failed to accumulate immediately in synaptic vesicles within isolated rat brain synaptosomal cytoplasm.

It was examined whether or not enzymically synthesized L-dopamine could be immediately incorporated and stored in synaptic vesicles within a synaptosomal cytoplasm fraction isolated from the rat brain. The synaptosomal cytoplasm was isotonically prepared by disrupting the synaptosomes in 0.32 M sucrose using an N2-gas pressure homogenizer. Synaptic vesicles present in the isolated synaptosomal cytoplasm appeared to be osmotically sensitive (i.e., not leaky vesicles) under iso-osmotic conditions. DOPA decarboxylase [EC 4.1.1.28] present in the soluble cytosol region synthesized L-[14C]dopamine from added L-[14C]DOPA. However, the newly synthesized L-[14C]dopamine was not immediately incorporated in the synaptic vesicles within the synaptosomal cytoplasm during the reaction period of 20 min at 30 degrees C. Plain synaptic vesicles prepared by the method of Kadota and Kadota (10) also failed to take up the newly synthesized dopamine under the same conditions. It was found that soluble protein(s) in the synaptosomal cytoplasm was able to bind as much added L-[3H]dopamine as 35 pmol per mg of protein, and the binding ability seemed to inhibit the amine uptake by synaptic vesicles within the synaptosomal cytoplasm. The [3H]dopamine bound to the protein(s) became unbound in the presence of Ca2+ (5 mM). This effect of Ca2+ may have an important role in the releasing process of neurotransmitters.

Animals↗

PubMed1993

Inhibition of dopamine transport in rat brain synaptosomes by volatile anesthetics.

BACKGROUND: Volatile anesthetics may depress transmission by altering synaptic concentrations of neurotransmitter. Microdialysis studies have found an increase in brain extracellular dopamine concentration on exposure to volatile anesthetics. We investigated the possibility that synaptosomal dopamine transport is reversibly inhibited by the halothane and isoflurane. METHODS: Rat brain synaptosomes were incubated with 5 nM 3H-DA and increasing concentrations of anesthetic in Teflon-sealed microvials. Cocaine (100 microM) was used to quantify non-specific binding/uptake. Uptake was stopped by vacuum filtration and washing; label incorporation into synaptosomes was determined by liquid scintillation counting. 3H-DA release from preloaded synaptosomes also was studied in the presence of the anesthetic to allow distinction between uptake inhibition and release stimulation in the synaptosomes. RESULTS: Both halothane and isoflurane inhibited the specific 3H-DA uptake in a concentration-dependent fashion with an IC50 of 0.72 +/- 0.14 mM for halothane and 2.24 +/- 0.85 mM for isoflurane. No stereoselectivity of isoflurane's action on Dopamine (DA) uptake was observed. The inhibition produced by halothane and isoflurane was kinetically characterized as noncompetitive, but full reversal was demonstrated after removal of the anesthetic from the incubation mixture. The anesthetics did not stimulate 3H-DA release from preloaded synaptosomes. CONCLUSIONS: These results demonstrate volatile anesthetic-induced inhibition of the dopamine transporter in this preparation of synaptosomes. The calculated IC50S suggest this inhibition occurs with clinically relevant concentrations of halothane but not with isoflurane. The results are consistent with and may explain the increase in extracellular dopamine concentrations demonstrated by microdialysis.

Animals↗

PubMed1981

A synaptosomal preparation from the guinea pig ileum myenteric plexus.

Our interest in investigating the presynaptic modulation of acetylcholine release led to the development of a synaptosomal preparation from the guinea pig ileum myenteric plexus-longitudinal muscle. A crude synaptosomal fraction (P2) was obtained by homogenization and differential centrifugation. The preparation exhibited a specific uptake system for choline and for noradrenaline (NA), but not for 5-hydroxytryptamine (5-HT). Synaptosomes were isolated from this P2 fraction by an isoosmotic density gradient prepared from sucrose and metrizamide. The resultant synaptosomal fraction was enriched about sevenfold in both choline uptake and in choline acetyltransferase (ChAT). Choline was transported by a high-affinity system was a Km of 6.5 X 10(-7) M and a Vmax of 41 pmol/mg protein/min. Electron microscopy confirmed the synaptosomal nature of the gradient fraction. Some synaptosomal profiles contained only small, translucent vesicles whereas others also contained large (approx. 100 nm diameter) electron-opaque vesicles. The crude synaptosomal fraction synthesized acetylcholine (ACh) from exogenous choline and it released the synthesized ACh in a calcium-dependent manner.

Acetylcholine↗

PubMed1983

Studies on the osmotic disruption and resealing of synaptosomes.

The release of lactate dehydrogenase and K+ when synaptosomes are exposed to resuspension in media of various osmolarity has been investigated in order to measure their disruption. Even when resuspended in distilled water a significant percentage (10-20%) of lactate dehydrogenase and K+ remains unreleased. The particles containing these substances sediment to the same density as synaptosomes. Synaptosomes retaining their internal organelles after hypoosmotic treatment can be seen in electron micrographs. Resealing of disrupted synaptosomes was measured by the inclusion of [14C]sucrose. The resealing is spontaneous, essentially complete (80-90%) within 20 min and not noticeably affected by temperature, pH, or the addition of fusogen. The synaptosome preparation after hypoosmotic disruption will therefore contain some undisrupted synaptosomes with some or all of their complement of cytoplasmic constituents, as well as resealed synaptosomes. The retention of the ability of the hypoosmotically treated preparation to convert [14C]choline to [14C]acetylcholine is demonstrated as an example of the disproportionate effect these undisrupted particles have on its properties.

Acetylcholine↗

PubMed1983

Metabolically active synaptosomes can be prepared from frozen rat and human brain.

Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue-derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1-5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter-related studies.

Animals↗

PubMed1983

Effect of liposomes containing various divalent cations on the release of acetylcholine from synaptosomes.

The effect of increasing the cytoplasmic levels of various divalent cations on the release of [3H]acetylcholine ([3H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [3H]choline were incubated with liposomes containing Mg2+, Ca2+, Mn2+, Co2+, Sr2+, or Ba2+. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [3H]ACh and [3H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg2+, Mn2+, or Co2+ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K+/Na+. Synaptosomes incubated with liposomes containing Ca2+, Sr2+, or Ba2+, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [3H]ACh. Liposomes containing either Ca2+ or Sr2+ were equally effective in promoting the release of [3H]ACh from synaptosomes, whereas liposomes containing Ba2+ were 2.5 times more effective in promoting the release of [3H]ACh than were liposomes containing either Ca2+ or Sr2+. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [3H]ACh caused by liposomes containing Ca2+, Sr2+, or Ba2+ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.

Acetylcholine↗

PubMed1988

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.

Adenosine Triphosphate↗

PubMed1989

ATP release, adenosine formation, and modulation of dynorphin and glutamic acid release by adenosine analogues in rat hippocampal mossy fiber synaptosomes.

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.

Adenosine↗

PubMed1989

Introduction of impermeant molecules into synaptosomes using freeze/thaw permeabilization.

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.

Acetylcholine↗