Depolarization-induced release of ATP from cholinergic synaptosomes is not blocked by botulinum toxin type A

We report here the effects of Botulinum Toxin type A on the release of ATP and Acetylcholine from Torpedo electric organ synaptosomes. Our results show that Botulinum Toxin type A inhibits specifically the K⁺-induced release of Acetylcholine from synaptosomes without affecting the release of ATP. Membrane potential and calcium uptake into cholinergic nerve terminals are not modified after Botulinum Toxin poisoning. It is suggested that either most of the ATP released during the depolarization of the cholinergic synaptosomes does not originate from cholinergic synaptic vesicles or that there are two populations of synaptic vesicles, Acetylcholine-enriched synaptic vesicles and ATP-enriched synaptic vesicles. However, the possibility that the ACh and ATP released could come from different intrasynaptosomal compartments cannot be excluded.     

Rearrangement of intramembrane particles as a possible mechanism for the release of acetylcholine

1. A chemiluminescent procedure for measuring acetylcholine (ACh) has recently been described. The procedure is based on the hydrolysis of ACh by acetylcholinesterase and on the oxidation of choline to betaine and H2O2 by choline oxidase. The H2O2 generated reacts with luminol in presence of peroxidase to produce a light emission. This method is sensitive in the pmol/ml range. 2. On isolated synaptosomes from electric organ, it is possible to obtain an estimate of the cytoplasmic ACh compartment by measuring the light emission after a single freezing and thawing cycle. The vesicular pool which resists several freezing and thawing cycles is then estimated by opening the compartment with a detergent. Increasing the intensity of stimulation of synaptosomes with different agents depletes the ACh content down to the vesicular pool. 3. The release of ACh is not associated with any change in the number of synaptic vesicles as seen in cryofractured synaptosomes. The only ultrastructural change detected common to all stimulations was a decreased density of P face intramembrane particles smaller than 11 nm and an increased density of E face 8 to 18 nm particles. The very significant particle changes were more intense for the conditions releasing more ACh. It is suggested that these particles are involved in the release of ACh from the cytoplasm. An attempt to directly correlate the release of ACh with intramembrane particle changes is discussed.     

Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.     

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.     

Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.     

Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

The action of botulinum toxin on cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata. Detection of a putative toxin receptor

Botulinum neurotoxin type A (BoNTx) inhibits the release of acetylcholine (ACh) from Torpedo electric organ synaptosomes. We have studied several biochemical and morphological aspects in order to characterize the molecular interactions of BoNTx intoxication in our preparation. 1. We are describing for the first time an electrophoretic band from cholinergic presynaptic plasma membrane (PSPM) that is recognized by 125I-BoNTx as a putative BoNTx receptor. 2. Furthermore we describe direct interaction of botulinum toxin-gold complexes with synaptic vesicles through the three-step model of the BoNTx intoxication.     

The subsynaptosomal distribution and release of [3H]acetylcholine synthesized by rat cerebral cortical synaptosomes

Synaptosomes were prepared from rat cerebral cortex and incubated in [³H]choline for periods ranging from 1 to 90 min. The [³H]ACh synthesized during this period was found only in the cytoplasm and in a membrane-associated fraction. A negligible amount of the newly formed [³H]ACh was recovered in the vesicular fraction despite concerted efforts to protect a hypothetical population of labile vesicles. The specific activity of the membrane-associated component, accounting for 21% of the total [³H]ACh, was by far the highest. This membrane-associated fraction was not released by hypotonic shock or homogenization and apparently was not in association with the monodisperse synaptic vesicles. The [³H]ACh was released in a calcium dependent manner. This investigation has determined that the ACh synthesized by synaptosomes is localized in only two fractions, cytoplasmic and membrane-associated; that this newly synthesized ACh can be released from synaptosomes by a process consistent with physiological release; and that at least part of the ACh released was originally present in the cytoplasm.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

Protein synthesis in synaptosomes: a proteomics analysis

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.     

The osmotically sensitive potassium and sodium compartments of synaptosomes

1. Synaptosomes are pinched-off nerve terminals whose components can be liberated by osmotic `shock'. A synaptosome preparation run through a Sephadex column that was eluted with an iso-osmotic solution retained its small ions, whereas when the column was eluted hypo-osmotically the small ions were lost. In this way the osmotically sensitive Na<sup>+</sup> and K<sup>+</sup> of synaptosomes were measured. Measurements of the lactate dehydrogenase occluded within the synaptosome were also made. The release of osmotically sensitive Na<sup>+</sup> and K<sup>+</sup> and occluded lactate dehydrogenase had similar characteristics with respect to the degree of osmotic `shock' necessary and the action of lytic agents. 2. The distribution of osmotically sensitive Na⁺, K⁺ and occluded lactate dehydrogenase in the subfractions of a crude mitochondrial preparation was examined. The synaptosome fraction was the richest source of these constituents. 3. On standing at 5° in iso-osmotic solution Na⁺ and K⁺ were lost from synaptosomes, whereas the amount of occluded lactate dehydrogenase remained stable, suggesting that the synaptosome membrane retained its integrity but that Na⁺ and K⁺ diffused through it out of the osmotically sensitive compartment. 4. The uptake of Na⁺ and K⁺ into the osmotically sensitive compartment was examined. At 5° the rates of uptake of Na⁺ and K⁺ were found to be equal to the rates of loss of these ions when correction to a uniform concentration gradient had been made. K⁺ travelled across the membrane slightly faster than Na⁺, the rate of K⁺ movement being about 1·0μμequiv.cm.⁻²sec.⁻¹ under a concentration gradient of 0·1m. Active transport is not thought to contribute to the ion movements under the conditions used. 5. The amount of K⁺ taken up into the osmotically sensitive compartment as a function of the external concentration was examined. Since the uncharged molecule d-[¹⁴C]galactose distributes across the synaptosome membrane similarly to K⁺ there is not thought to be a synaptosomal trans-membrane potential. The volume of the osmotically sensitive compartment was measured by this method and found to agree with estimates of the synaptosomal volume made from morphological studies. In media of low ionic strength synaptosomes exhibit a Donnan effect. 6. It is concluded from these studies that the osmotically sensitive compartment represents the inner volume of the synaptosome, which is completely separated from the outside environment by a diffusion barrier having many of the general properties of a biological membrane.     

Stimulation of the production of unesterified fatty acids in nerve endings of guinea-pig brain in vitro by noradrenaline and 5-hydroxytryptamine

1. Noradrenaline (1mm) and 5-hydroxytryptamine (1mm) stimulated the production of unesterified palmitate, oleate, stearate and arachidonate in nerve endings (synaptosomes) isolated from combined guinea-pig cerebral cortex and cerebellum. 2. Iproniazid phosphate (0.36mm) increased the concentrations of the same acids in osmotically ruptured synaptosomes. Further addition of 1mm-noradrenaline or 1mm-5-hydroxytryptamine reversed this increase. 3. Noradrenaline (0.01mm) stimulated the production of unesterified fatty acids in isolated synaptic membranes. 5-Hydroxytryptamine (0.01mm) stimulated the production of unesterified fatty acids in synaptic membranes and synaptic vesicles.     

THE ISOLATION OF CHOLINERGIC SYNAPTIC VESICLES FROM BOVINE SUPERIOR CERVICAL GANGLION AND ESTIMATION OF THEIR ACETYLCHOLINE CONTENT

Abstract— A method is described for the isolation of relatively pure cholinergic synaptic vesicles by hypo-osmotic shock and density gradient centrifugation of a synaptosome fraction prepared from bovine superior cervical ganglia. Vesicles from this source were found to sediment to a density equivalent to 0·3–0·41 M-sucrose. The vesicle subfraction from the gradient had an acetylcholine (ACh) content of 4.4 nmol/mg of protein and were subject to leakage of ACh. By a 'tagging' technique, the vesicles were counted under the electron microscope and their numbers related to their ACh concentration. After correction for leakage, an ACh content of 1630 molecules/vesicle was estimated.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

ISOLATION OF [3H]ACETYLCHOLINE POOLS BY SUBCELLULAR FRACTIONATION OF CEREBRAL CORTEX SLICES INCUBATED WITH [3H]CHOLINE

Abstract— Subcellular fractions were isolated from tissue incubated in [³H]choline with or without the addition of 33 mM-KCl. Radioactive and bioassayable ACh were measured in the synaptosomes, synaptosomal cytoplasm and in the vesicles. After incubation with KCI the vesicles, as isolated, contained ACh of a lower specific activity than the cytoplasmic ACh. Therefore the vesicle fraction as isolated does not represent the source of the high specific activity ACh released upon K⁺ stimulation. However the vesicle fraction is heterogeneous. Most of the bioassayable ACh but little of the radioactive ACh in the vesicles passed through iso-osmotic Sephadex columns. These results raise the question of the existence of vesicles which contain highly radioactive ACh but which lose it during their isolation by current methods. Different possible forms of heterogeneity are discussed.     

SUBMICROSCOPIC CHANGES OF THE SYNAPSE AFTER NERVE SECTION IN THE ACOUSTIC GANGLION OF THE GUINEA PIG. AN ELECTRON MICROSCOPE STUDY

The degenerative changes of the synaptic regions after nerve section have been studied with the electron microscope in the interneuronal synapse of the ventral ganglion of the acoustic nerve of the guinea pig. Fixation with buffered osmic tetroxide was carried out 22, 44, and 48 hours after destruction of the cochlea on one side; the contralateral ganglion being used as control. The submicroscopic organization of normal axosomatic and axodendritic synapses is described. In the synaptic ending four morphological components are recognized: the membrane, the mitochondria, the synaptic vesicles (19, 20), and the cytoplasmic matrix. The intimate contact of glial processes with the endings and with the surface of the nerve cell is described. At the level of the synaptic junction there is a direct contact of the limiting membranes of the ending and of the cell body or dendrite. Both contacting membranes constitute the synaptic one with a total thickness of about 250 A. This membrane has regions of higher electron density where the synaptic vesicles come into intimate contact and fuse with it. Definite degenerative submicroscopic changes in the nerve endings were observed after 22 hours of destruction of the cochlea and were much more conspicuous after 44 and 48 hours. After 22 hours there is swelling of the ending and decreased electron density of the matrix. Most synaptic vesicles have disappeared or seem to undergo a process of clumping and dissolution. Some mitochondria also show signs of degeneration. After 44 hours the synaptic vesicles have practically disappeared; mitochondria are in different stages of lysis; the membrane of the ending becomes irregular in shape, and there is shrinkage and in some cases detachment of the ending. No changes in the postsynaptic cytoplasm were observed. These observations and particularly the rapid lysis of the synaptic vesicles are discussed in correlation with data from the literature indicating the early alteration of synaptic function and the biochemical changes occurring after section of the afferent nerve. The hypothesis that the synaptic vesicles may be carriers of acetylcholine or other active substances (19, 20) and that they may act as biochemical units in synaptic transmission is also discussed.²     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.