The release of acetylcholine: from a cellular towards a molecular mechanism

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge. Consequently, the mechanism releasing ACh from the cytoplasm in a packet form was searched at the presynaptic membrane itself. With synaptosomes isolated from the electric organ of Torpedo, it became possible to freeze them rapidly at the peak of ACh release and study their membrane and contents after cryofracture. A statistical analysis showed that the main structural change was the occurrence of large intramembrane particles at the peak of ACh release and under all release conditions. This impressive change contrasted with the stability in the number of vesicles. Another role for the vesicle was envisaged during intense stimulations when the cytoplasmic ACh and ATP pools become exhausted. The decrease in ATP leads to an increase in calcium and protons in the cytoplasm; this signals the depletion of vesicular ACh and ATP stores in the cytoplasm. Release can go on, while ATP promotes the uptake of calcium by vesicles. At the end of its cycle the vesicle will be full of calcium and will perhaps release it. As far as the mechanism of ACh release is concerned it probably depends on a membrane component (perhaps the large particles) activated by calcium and able to translocate ACh in a quantal or subquantal form. In most recent work we showed that if a lyophilized presynaptic membrane was used to make proteoliposomes filled with ACh, they released ACh upon calcium action.     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of 2-(4-Phenylpiperidino)cyclohexanol on Acetylcholine Release and Subcellular Distribution in Rat Striatal Slices

These experiments measured the effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of acetylcholine (ACh) and its subcellular distribution in slices of rat striatum incubated in vitro. The AH5183, a drug that blocks the uptake of ACh by isolated synaptic vesicles, reduced the release of ACh from slices stimulated to release transmitter in response to K+ depolarization. Tissue stimulated in the presence of AH5183 contained more ACh in a nerve terminal cytoplasmic fraction than did tissue stimulated in the drug's absence, but stimulation in AH5183's presence reduced the amount of ACh measured in fractions containing synaptic vesicles. The depletion of ACh caused by stimulating tissue in the presence of AH5183 was more evident in the fraction of nerve terminal ACh occluded within synaptic vesicles as isolated by gradient centrifugation (fraction D) than it was in other nerve terminal occluded stores. It is concluded that the synaptic vesicles isolated as fraction D under the present experimental conditions likely contain releasable transmitter. The AH5183 also depressed the spontaneous release of ACh from incubated slices of striatum and this effect was evident in the presence or the absence of medium Ca2+. It is suggested that this effect might indicate that the process of spontaneous ACh release measured neurochemically results, in part, from an AH5183-sensitive carrier-mediated process.     

Mobilization of a Vesamicol-Insensitive Pool of Acetylcholine from a Sympathetic Ganglion by Ouabain

Abstract: These experiments investigate the release of transmitter from the perfused superior cervical ganglia of cats induced by ouabain in the absence or presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a blocker of acetylcholine (ACh) uptake. Ouabain, perfused through the ganglia, released ACh in a Ca²⁺-dependent way. Vesamicol caused some inhibition of the release of ACh by ouabain; however, under this condition, the Na⁺, K⁺-ATPase inhibitor released five times more transmitter than did preganglionic stimulation at 5 Hz. Also, when ganglia exposed to vesamicol were depleted of the impulse-releasable pool of ACh, subsequent perfusion with ouabain released ACh, and this included ACh newly synthesized in the presence of vesamicol; this phenomenon could be inhibited by the lack of Ca²⁺ and presence of EGTA, and was completely abolished by perfusion with a medium containing 18 mM Mg²⁺. To test whether the release of this vesamicol-insensitive Ca²⁺-dependent pool by ouabain is associated with a decrease in the number of synaptic vesicles, ganglia treated with the ATPase inhibitor after the depletion of the impulse-releasable pool of ACh were fixed for electron microscopy. In the presence of Ca²⁺, coincident with the release of the vesamicol-insensitive pool of ACh, nerve terminals were almost depleted of synaptic vesicles; ganglia treated similarly, but with medium containing 18 mM Mg²⁺ instead of Ca²⁺, were not depleted of synaptic vesicles. These results suggest that ouabain releases a vesamicol-insensitive pool of ACh from the sympathetic ganglion and also support the notion that this compartment is vesicular and its exocytosis depends on extracellular Ca²⁺. It is suggested that empty-vesicle recycling in the presence of vesamicol restricts mobilization of full vesicles to release sites.     

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.     

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.     

Exocytotic Release of [3H]-Acetylcholine by Ouabain Involves Intracellular Ca2+ Stores in Rat Brain Cortical Slices

1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of [3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of [3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase [3H] ACh exocytosis from rat brain cortical slices.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

Biogenesis of synaptic vesicles in vitro

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.     

Choline analogues: their use in studies of acetylcholine synthesis, storage, and release

The objective of this article is to illustrate how choline analogues might provide insight into mechanisms that regulate the synthesis, storage, and release of acetylcholine (ACh). Studies with false neurotransmitters provide information about the origin of releasable transmitter. Thus, false esters that distribute like ACh to vesicle-bound stores are as releasable as is ACh, but esters that poorly localize to synaptic vesicles are poorly releasable. Studies of choline analogue uptake provide information about the structural specificity of that transport process and, also, show that choline uptake is regulated in response to activity. Thus, stimuli that normally release transmitter increase the rate of choline transport, presumably to provide more precursor for ACh synthesis. However, the relationship between precursor delivery and product formed can be dissociated, suggesting that some factor in addition to choline delivery is involved in ACh synthesis regulation. Studies with a compound (AH5183), which inhibits ACh uptake by synaptic vesicles, provide information about the relationship of ACh stores and releasable transmitter. In the presence of AH5183 some 15% of nerve terminal ACh is released in response to nerve impulses, suggesting the existence of a small population of vesicles that contain readily releasable ACh. In presence of AH5183, ACh synthesis is activated even when ACh release is depressed, showing that transmitter synthesis can be regulated by some factor other than nerve terminal ACh levels.     

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.     

Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.     

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles.

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [³²p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.     

A simple model for calcium induced exocytosis

We have developed a simple model showing how the presence or absence of Ca²⁺ can determine whether an uncurved or curved membrane surface is favored energetically. The model shows why fusion of vesicles with the presynaptic membrane is favored in the presence of calcium and why the budding off of vesicles is favored in the absence of calcium inside of the presynaptic membrane. The model accurately predicts the radius of a synaptic vesicle using known properties of lipids and suggests consequences of temperature change, varied stimulation rate and addition of calcium by artificial means on rates of transmitter release.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

硝苯吡啶对腹腔神经节突触传递的阻断作用

本文应用细胞内记录技术,观察了钙通道阻滞剂硝苯吡啶（nifedipine)对离体豚鼠腹腔神经节突触传递的影响,硝苯吡啶（0.1-10umol/L)不影响所检细胞的静息膜电位,膜电阻及细胞内刺激引起的动作电位,但能显著阻断N-型胆碱能的突触传递,并且这种作用可被低钙模拟、高钙拮抗,硝苯吡啶（10umol/L)也不影响突触后膜对乙酰胆碱（ACh)的敏感性;但在高钾克氏液中,能减少微小兴奋性突触后电位（mEPSPs)的频率;在低钙和高镁克氏液中,能减少量子含量,而对量子大小无影响。结果表明,治疗量的硝苯吡啶(0.1umol/L)通过阻滞突触前膜钙内流及ACh的量子性释放,产生突触阻断作用。这可能是硝苯吡啶降压机理的一个组成部分。     

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.     

Ouabain evokes exocytosis dependent on ryanodine and mitochondrial calcium stores that is not followed by compensatory endocytosis at the neuromuscular junction

Ouabain is a cardiotonic glycoside that inhibits the sodium potassium ATPase pump leading to sodium accumulation in nerve terminals. At the frog neuromuscular junction, ouabain induces acetylcholine release and a rapid depletion of synaptic vesicles. In the present work, we used FM1–43 vital labeling to dissect the effect of ouabain on synaptic vesicles recycling. We first examined images of nerve-muscle preparations that were stained with FM1–43 by electrical stimulation of the nerve and destained with ouabain. We observed that ouabain induced exocytosis of synaptic vesicles independently of extracellular calcium, implying a mechanism of exocytosis that can bypass the requirement for extracellular calcium. We therefore tested the hypothesis that ouabain induces exocytosis by mobilizing intracellular calcium and we report that calcium release from endoplasmic reticulum through ryanodine receptors is necessary for ouabain-evoked exocytosis. In addition, the ouabain-evoked exocytosis was dependent on calcium released from mitochondria. We also investigated if exocytosis evoked by ouabain is followed by compensatory endocytosis. We observed that muscles incubated with FM1–43 in the presence of ouabain did not present significant staining. In conclusion, our data demonstrate that exocytosis evoked by ouabain is independent on extracellular calcium but dependent on calcium release from endoplasmic reticulum and mitochondrial stores. In addition, we suggest that ouabain can be used as a pharmacological tool to uncouple synaptic vesicles exocytosis from endocytosis at the neuromuscular junction.