AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways

In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+-dependent K+-mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.     

Binding and Active Transport of Large Analogues of Acetylcholine by Cholinergic Synaptic Vesicles In Vitro

A previous structure-activity investigation of acetylcholine (ACh) revealed a positive correlation between additional hydrophobic bulk and increased potency for inhibition of active transport of [3H]ACh by synaptic vesicles isolated from the electric organ of Torpedo. In the current study, several ACh analogues that are significantly larger than previously studied "false transmitters" were synthesized in the tritiated form by chemical means and tested for active transport. These are analogue 14 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-acetoxypyrrolidinium iodide], analogue 15 [(+/-)-1,1-dimethyl-3-benzoyloxypyrrolidinium iodide], and analogue 16/17 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-benzoyloxypyrrolidinium iodide]. These analogues place significant additional hydrophobic bulk on one or the other (analogues 14 and 15) or both (analogue 16/17) of the two pharmacophores of a small, conformationally constrained analogue of ACh. [3H]Analogue 14 and [3H]analogue 15 are actively transported, with Vmax values the same as or less than that of ACh, depending on the vesicle preparation. The observation that Vmax is the same for an analogue and ACh in some vesicle preparations suggests that the rate-limiting step does not involve ACh bound to the transporter. [3H]Analogue 16/17 is actively transported very poorly. Km values for ACh and for transported ACh analogues vary by up to two- to threefold in different vesicle preparations. The ACh transporter is much less selective for transported substrates than anticipated.     

Mg-ATPase and Torpedo Cholinergic Synaptic Vesicles

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.     

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.     

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.     

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.     

Postnatal Changes in Enzyme Activities in Synaptosomes Isolated from Hamster Optic Nerve Endings

Subsynaptosomal fractions isolated from optic terminal nuclei of adult and neonatal hamsters exhibited developmental changes in specific density, mitochondrial activity, and K+-stimulated, ouabain-inhibited p-nitrophenylphosphatase (K-pNPPase) activity around the time of eye opening. The specific activity of K-pNPPase was six- to sevenfold higher after eye opening (14-16 days postnatal). A significant proportion of high-specific- activity K-pNPPase was recovered from the lightest subsynaptosomal fraction at all ages. This fraction contained very little external membrane by galactose oxidase - NaB3H4 labeling, suggesting that it may represent an internal pool, possibly the axonally transported form of the enzyme. Synaptic mitochondrial cytochrome c. oxidase activity also approximately doubled in the period between 12 and 16 days. The specific density of the external membrane increased very slowly, banding at 1.0 M sucrose at 12 and 16 days, and at 1.2 M in adults. These maturational events may reflect increased energetic needs for optic nerve endings following eye opening.     

Cholinergic Modulation of the Release of [3H]Acetylcholine from Synaptosomes of the Myenteric Plexus

Abstract: The purpose of these experiments was to determine if cholinergic agents affected the release of acetylcholine (ACh) from a synaptosomal preparation of the guinea pig ileum myenteric plexus. The synaptosomal preparation was first incubated with the precursor [³H]choline; subsequently, release of the stored [³H]ACh was measured. The release was decreased by oxotremorine or exogenous ACh plus hexamethonium and increased by exogenous ACh plus atropine. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) evoked release that was inhibited by nicotinic antagonists or muscarinic agonists. Release was stimulated half-maximally by approximately 2 μ m - and maximally by 10 μ m -DMPP. Either in the absence of calcium or at 0°C, DMPP was without effect. The effect of 10 μ m -DMPP was brief, a significant stimulation occurring only within the first 2 min at 37°C. Tetrodotoxin also inhibited excitation by DMPP but not completely. Thus, the release of [³H]ACh appears to be presynaptically modulated, negatively by muscarinic agonists and positively by nicotinic agonists.     

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.     

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.     

Ultrastructural Changes Induced by 12-O-Tetradecanoylphorbol 13-Acetate in Pure Cholinergic Synaptosomes of Torpedo Electric Organ

We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.     

Acetylcholine Turnover and Compartmentation in Rat Brain Synaptosomes

Abstract: The turnover of acetylcholine (ACh) in rat brain synaptosomes and its compartmentation in the labile bound and stable bound pools were investigated. The P₂ fraction from rat brain was subjected to three sequential incubations, each terminated by centrifugation followed by determination of ACh concentrations by gas chromatography-mass spectrometry (GCMS): (1) Depletion phase: Incubation of synaptosomes at 37°C for 10 min in Na⁺-free buffer containing 35 mM-KCl reduced the content of both labile bound and stable bound ACh by 40%. (2) Synthesis phase: Incubation at 37°C with 2 μ M -[²H₄]choline resulted in accumulation of labeled and unlabeled ACh in both compartments. Addition of an anticholinesterase had little effect on stable bound ACh but greatly increased the content of labile bound ACh. This excess accumulated ACh was probably due to inhibition of intracellular acetylcholinesterase (AChE), because negligible uptake of ACh from the medium was observed. The effects on ACh synthesis of altered cation concentrations and metabolic inhibitors were examined. (3) Release phase: The tissue was incubated in the presence of 35 mM-KCl, 40 μM-paraoxon, and 20 μM-hemicholinium-3 (HC-3) (to inhibit further synthesis of ACh). Measurements of the compartmental localization of ACh at several time points indicated that ACh was being released from the labile bound fraction. In support of this conclusion, 20 mM-Mg²⁺ reduced ACh release and increased the labile bound ACh concentration.     

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.     

Calmodulin Binding Proteins of the Cholinergic Electromotor Synapse: Synaptosomes, Synaptic Vesicles, Receptor-Enriched Membranes, and Cytoskeleton

Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs--Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane-associated. The Triton X-100-insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin-coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.     

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.     

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.     

Release of Acetylcholine from Rat Brain Synaptosomes Stimulated with Leptinotarsin, A New Neurotoxin

Abstract: Leptinotarsin is a neurotoxic protein found in the hemolymph of potato beetles of the genus Leptinotarsa. In order to study the action of leptinotarsin from two species, L. haldemani and L. decemlineata , synaptosomes were prelabeled with [³H]choline in order to synthesize [³H] acetylcholine (ACh). These synaptosomes were then immobilized on Millipore filters and used for assay. Toxins from both species induce the release of radioactivity in this system. Fractionation of the released radioactivity indicated that ACh was released in preference to choline. The toxin that caused release was heat-labile and was partially dependent on Ca²⁺ in the perfusing medium. Release followed apparent first order kinetics when stimulation was effected with leptinotarsin from L. haldemani (leptinotarsin-h), but was more complex when using leptinotarsin from L. decemlineata (leptinotarsin-d). Increasing the concentration of toxin increased the rate of release, but the shapes of the dose-release curves elicited by the leptinotarsins from the two species were different. While leptinotarsin-h exhibited a simple, saturating dose-release curve, leptinotarsin-d was characterized by a sigmoid function, which was well described, with a Hill coefficient of 1.8. Antibodies directed toward black widow spider venom glands had no effect upon the releasing activity of leptinotarsin-h but could partially neutralize that of leptinotarsin-d. Toxins from both species have been partially purified and do not appear to be identical. The purified toxins should be useful tools with which to study the release of acetylcholine.     

Uncoupling of Cholinergic Synaptic Vesicles by the Presynaptic Toxin β-Bungarotoxin

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.     

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.