Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.     

Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.     

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.     

Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze-fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Effect of the venom of Glycera convoluta on the spontaneous quantal release of transmitter

A neurotoxin able to increase the spontaneous release of transmitter was found in the venom glands of the polychaete annelid Glycera convoluta. We studied the effect of this venom on the frog cutaneous pectoris muscle, where its application produced a prolonged (20-h), high-frequency discharge of miniature potentials. After 5 h of action, the initial store was renewed several times but no detectable ultrastructural changes were observed. After 19 h of sustained activity, nerve terminals with their normal vesicular contents were infrequent; others were fragmented and contained swollen mitochondria, abnormal inclusions, and vesicles of various sizes. In the noncholinergic crayfish neuromuscular preparation, the venom triggered an important increase in spontaneous quantal release that subsided in 1 h. An activity higher than that in resting conditions then persisted for many hours. This high electrical activity was not accompanied by any detectable structural modifications after 3 h. In the torpedo electric organ preparation, the venom elicited a burst of activity that returned to control levels in 1 h. The release of ACh (evaluated by the efflux of radioactive acetate) paralleled the high electrical activity. No morphological changes or significant depletion of tissue stores were detected. The venom of Glycera convoluta appears to enhance considerably the release of transmitter without impairing its turnover. The venom effect is Ca++ dependent and reversible by washing, at least during the first hour of action. Because the high rate of transmitter release appears dissociated from the later-occurring structural modifications, it is possible that the venom mimics one component of the double mode of action proposed for black widow spider venom.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

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.     

Large-Scale Purification of Torpedo Electric Organ Synaptosomes

Abstract: A procedure for the large-scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium. Two hundred grams of electric organ can be fractionated in a single run, allowing biochemical studies on presynaptic membrane constituents.     

Botulinum neurotoxin inhibits the release of newly synthesized acetylcholine from torpedo electric organ synaptosomes

In previous reports, we have shown that botulinum neurotoxin inhibits acetylcholine release from Torpedo marmorata electric organ and from its synaptosomal fraction. Here, we have focussed our attention on the study of the effect of botulinum neurotoxin on the metabolism of acetylcholine, namely, the precursors supply, the synthesis activity and the storage of the neurotransmitter into nerve endings isolated from Torpedo electric organ. Radiolabelled acetylcholine precursors (acetate and choline) uptake, choline O-acetyltransferase activity, and the compartmentalization of the transmitter into the synaptosomes were not modified by botullinum neurotoxin. When labelled nerve ending were depolarized by K⁺, the specific radioactivity of acetylcholine in the free pool fell markedly, but the specific radioactivity in the bound pool remained constant. Botulinum neurotoxin prevented this K⁺-induced decrease of specific radioactivity in the free pool.     

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1–8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1–8 monoclonal antibody, it was possible to show that they are different proteins. The C1–8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1–8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1–8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Postsynaptic effects of crotoxin and of its isolated subunits.

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.     

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Ouabain induces acetylcholine release from pure cholinergic synaptosomes independently of extracellular calcium concentration

We have studied the correlation between [³H]ouabain binding sites, (Na⁺+K⁺)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions ofTorpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [³H]ouabain binding sites in good agreement with the high (Na⁺+K⁺)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca²⁺ _o may be an increase in Na⁺ _i that could (a) evoke Ca²⁺ release from internal stores and (b) inhibit ATP-dependent Ca²⁺ uptake by synaptic vesicles.     

Structural changes at pure cholinergic synaptosomes during the transmitter release induced by A-23187 inTorpedo marmorata

Summary Pure cholinergic synaptosomes isolated from the electric organ ofTorpedo marmorata were stimulated by calcium ionophore A-23187. The effect of time course of stimulation on the changes in intramembrane particles (IMPs) on presynaptic membranes was studied by quickfreezing and aldehyde-fixation freeze-fracture. We showed that the decrease of small-particle density at the P-face and the increase of large-particle density at the E-face was maximum after 30 sec of A-23187 stimulation. Later, the density of synaptic vesicles decreased. We suggest that the redistribution of IMPs on the presynaptic membrane and acetylcholine (ACh) release from pure cholinergic synaptosomes have a similar time course when triggered by A-23187     

A Monoclonal Antibody Raised Against Plasma Membrane of Cholinergic Nerve Terminals of the Torpedo Inhibits Choline Acetyltransferase Activity and Acetylcholine Release

Monoclonal antibodies were raised against the synaptosomal plasma membranes (SPMs) purified from the electric organ of the Torpedo. One antibody that reacts preferentially with SPMs rather than with other membrane fractions isolated from this tissue was previously found to inhibit hydrophilic and amphiphilic choline-O-acetyltransferase (ChAT) activity. On immunoblots of SPMs, this antibody recognizes two polypeptides of 135 and 66 kilodaltons that are related; the 66-kilodalton polypeptide appears to exist as a monomer and as a dimer in SPMs. The antibody was also able to inhibit the calcium-dependent release of acetylcholine in Torpedo synaptosomes without affecting the total neurotransmitter content. This inhibition was dependent on the antibody concentration and was observed when the release was elicited by either KCl depolarization or the calcium ionophore A23187; this suggests that inhibition was not mediated by a blockage of the depolarization-activated calcium influx. The inhibition could not be prevented by atropine, a result indicating that the antibody does not block release by mimicking the action of acetylcholine on presynaptic muscarinic autoreceptors. Thus, the antigen recognized by this antibody appeared to be involved in acetylcholine release; this antigen could be membrane-bound ChAT, another protein of the SPMs, or both.     

ASSOCIATION OF SYNTAXIN WITH SNAP 25 AND VAMP (SYNAPTOBREVIN) IN TORPEDO SYNAPTOSOMES

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2–5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H⁺ ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses. Copyright © 1996 Elsevier Science Ltd     

Calcium-Dependent and -Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

Abstract: The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 × 10⁻⁷ M ), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 × 10⁻⁷ M to 10⁻⁵ M , the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10⁻⁶ M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations ( 10⁻⁵ M ), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.