Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca²⁺-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [³H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.     

Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."     

Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

Botulinum toxin a inhibits acetylcholine release from cultured neurons in vitro

Summary Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions. However, the detailed mechanism of this effect remains elusive. One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system. We studied the clonal pheochromocytoma PC12 cell line to establish such a model. PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties. Stimulation of these cells with high K⁺ (80 mM) in the perfusion medium markedly increased calcium-dependent [³H]ACh release compared to undifferentiated cells. Stimulated [³H]ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h. BoTx inhibition of [³H]ACh release was time- and concentration-dependent. A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration. The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated [³H]ACh release was 30 min. Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect. Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release. This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.     

Characterization of acetylcholine release from insect synaptosomes

Synaptosomes prepared from ganglia of Locusta migratoria, are able to accumulate [³H]choline and convert most of it to acetylcholine. Exposure of the labelled synaptosomes to media containing elevated K⁺ concentrations evoked a large increase in the efflux of tritiated acetylcholine. Some characteristics of acetylcholine release from insect nerve terminals were studied by continously perfusing synaptosomes with various solutions. Depolarization of the nerve endings with veratridine or K⁺ induced a release which was dependent on extracellular calcium, whereas Mg²⁺ inhibited the release. Pretreatment with the Ca²⁺-ionophore, A 23187, allowed a calcium-induced release under non-depolarizing conditions. The calcium-dependent efflux is thought to reflect stimulus-secretion coupling processes. In the presence of eserine and carbamylcholine the release was inhibited. Analysis of various cholinergic drugs revealed that the evoked efflux was susceptible to muscarinic ligands, it was enhanced by atropine and reduced by oxotremorine. The results suggest a feed-back regulation of acetylcholine release via muscarinic autoreceptors.     

Muscarinic receptors modulating acetylcholine release from insect synaptosomes

1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.     

Seasonal variations in the muscarinic regulation of acetylcholine release from Torpedo electric organ nerve terminals

The cholinergic nerve endings of the electric organs of Torpedo ocellata contains presynaptic muscarinic acetylcholine receptors (mAChR) which regulate acetylcholine (ACh) release by negative feedback. The efficiency of this muscarinic regulation varies circannually: maximal inhibition is observed in the winter, much smaller effects in the fall and spring, and no effect is observed during the summer. These variations are accompanied by seasonal changes in the ability of the mAChR to trigger the synthesis of its second messenger (a prostaglandin E-like substance) and in the ability of exogenous prostaglandin E2 to inhibit ACh release. No seasonal changes were found in the number of presynaptic mAChRs. These findings suggest that the observed seasonal variations are due to changes in both the metabolism of prostaglandins in the electric organ and the sensitivity of the ACh-releasing apparatus to the muscarinic second messenger.     

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).     

Effects of vesamicol on acetylcholine metabolism and synaptic transmission in the electric organ of Torpedo

Vesamicol [2-(4-phenylpiperidino)cyclohexanol, formerly AH5183] at a concentration of 10 μM reduced by 16–20% the amount of vesicle-bound ACh in intact pieces of Torpedo electric organ (isolated prisms). When [¹⁴C]acetate was applied to prisms in the presence of 10 μM vesamicol, vesicular translocation of newly synthesized [¹⁴C]ACh was inhibited by 40%. During short trains of field shocks given at 10 Hz to the tissue, vesamicol inhibited by 93% the release of [¹⁴C]ACh, but left the release of prestored ACh unaltered. In spite of these alterations, 10 μM vesamicol did not impair nerve-electroplaque transmission, even after prolonged electrical stimulation and during a recovery period. It is concluded that in the Torpedo electric organ the actions of vesamicol on ACh metabolism have apparently little or no effect on the efficiency of synaptic transmission.     

Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons

We examined effects of botulinum neurotoxin A (BoNTA) on sympathetic constrictions of the vena cava and uterine artery from guinea pigs to test the role of soluble NSF attachment protein receptor (SNARE) proteins in release of the cotransmitters norepinephrine (NE) and neuropeptide Y (NPY). Protein extracts of venae cavae and uterine arteries showed partial cleavage of synaptosomal associated protein of 25 kDa (SNAP-25) after treatment in vitro with BoNTA (50-100 nM). The rising phase of isometric contractions of isolated venae cavae to field stimulation at 20 Hz, mediated by NE acting on alpha-adrenoceptors, was reduced significantly by 100 nM BoNTA. However, sustained sympathetic contractions mediated by NPY were not affected by BoNTA. In uterine arteries, noradrenergic contractions to 1-Hz stimulation were almost abolished by BoNTA, and contractions at 10 Hz were reduced by 50-60%. We conclude that SNARE proteins are involved in exocytosis of NE from synaptic vesicles at low frequencies of stimulation but may not be essential for exocytosis of NPY and NE from large vesicles at high stimulation frequencies.     

Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells

This study investigated the presence of cell membrane docking proteins synaptosomal‐associated protein, 25 and 23 kD (SNAP‐25 and SNAP‐23) in satellite glial cells (SGCs) of rat trigeminal ganglion; whether cultured SGCs would release glutamate in a time‐ and calcium‐dependent manner following calcium‐ionophore ionomycin stimulation; and if botulinum neurotoxin type A (BoNTA), in a dose‐dependent manner, could block or decrease vesicular release of glutamate. SGCs were isolated from the trigeminal ganglia (TG) of adult Wistar rats and cultured for 7 days. The presence of SNAPs in TG sections and isolated SGCs were investigated using immunohistochemistry and immunocytochemistry, respectively. SGCs were stimulated with ionomycin (5 μM for 4, 8, 12 and 30 min.) to release glutamate. SGCs were then pre‐incubated with BoNTA (24 hrs with 0.1, 1, 10 and 100 pM) to investigate if BoNTA could potentially block ionomycin‐stimulated glutamate release. Glutamate concentrations were measured by ELISA. SNAP‐25 and SNAP‐23 were present in SGCs in TG sections and in cultured SGCs. Ionomycin significantly increased glutamate release from cultured SGCs 30 min. following the treatment (P < 0.001). BoNTA (100 pM) significantly decreased glutamate release (P < 0.01). Results from this study demonstrated that SGCs, when stimulated with ionomycin, released glutamate that was inhibited by BoNTA, possibly through cleavage of SNAP‐25 and/or SNAP‐23. These novel findings demonstrate the existence of vesicular glutamate release from SGCs, which could potentially play a role in the trigeminal sensory transmission. In addition, interaction of BoNTA with non‐neuronal cells at the level of TG suggests a potential analgesic mechanism of action of BoNTA.     

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.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Transient release of acetylcholine from Torpedo synaptosomes in response to prolonged depolarization

The release of ACh (acetylcholine) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay. A maintained depolarization by high KCl in the presence of Ca2+ triggered only a transient ACh release. It was shown that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response. The termination of release was probably caused by inactivation of voltage-dependent Ca2+ entry and rapid removal of intraterminal Ca2+ by a (Na+)0 dependent mechanism. It was found that exposure of the synaptosomes for a short period to low Ca2+-high K+ solutions greatly reduced the responses to Ca2+ reintroduction, as compared to the control release obtained when high K+ was applied in the presence of normal Ca2+. The response to Ca2+ reintroduction was measured following various times of preincubation with high K+ and low Ca2+; thus, an estimate of the time course of the inactivation of Ca2+ permeability during a depolarization could be made. A two component exponential kinetic was observed, with a rapid (tau = 3.6 s) and a slow phase (tau = 77 s). This inactivation was more pronounced when a higher KCl concentration was used to induce a greater depolarization. The presence of EGTA during the preincubation with high KCl greatly increased the response provoked by Ca2+ reintroduction, whereas increases in Ca2+ during the preincubation period caused proportional reduction in the subsequent response to Ca2+ reintroduction, indicating that the Ca2+ influx itself was involved in the inactivation process.     

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.     

Characterization of newly synthesized proteoglycans from rabbit menisci in organ culture.

Rabbit menisci were incubated with Na2 35SO4 in short-term organ culture to label newly synthesized proteoglycans. The radioactive products present in both tissue and culture medium were characterized separately with respect to distribution after ultracentrifugation in CsCl isopycnic density gradients, hydrodynamic size, interaction with hyaluronic acid, and glycosaminoglycan composition (types, size and content). Analysis of proteoglycan size by gel-filtration chromatography of the most-dense CsCl fractions (A1) on Sephacryl S-500 (associative conditions) resolved three species. A peak with Kav. approx. 0.7 was present in each chromatogram, and constituted the principal component in tissue extracts. Two other peaks with Kav. values of approx. 0.2 and 0.45 were also found. When the A1 fraction from tissue was subjected to CsCl-density-gradient ultracentrifugation under dissociative conditions, 71% of the recovered radioactivity was present in the most dense (A1D1) fraction. Incubation with hyaluronic acid of either A1 or A1D1 fraction from associative extract did not alter the apparent size of the labelled product, indicating a lack of aggregate formation. Meniscal proteoglycans showed an unusual and marked tendency to adsorb irreversibly to agarose and agarose-containing gel-filtration-chromatography media. High-pressure liquid-chromatographic analyses indicated that the sulphated glycosaminoglycans consisted of chondroitin 6-sulphate (72%), chondroitin 4-sulphate (19%) and dermatan sulphate (5%). Endo-beta-galactosidase (keratanase) digestion of the material failed to detect the presence of keratan sulphate. Of the labelled glycosaminoglycans, 95% was eluted from Sephacryl S-400 as a single symmetrical peak with a Kav. of 0.5. The results of studies with tissue extracts and culture medium were similar.     

Evaluation of acetate uptake and its relative conversion to acetylcholine in Torpedo electric organ synaptosomes under different ionic and metabolic conditions

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10^?4 M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis.Acetate uptake displayed a marked requirement for external Na⁺ and was decreased following depolarization of the synaptosomes by an elevated K⁺ concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca²⁺, while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself.The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.