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).     

Neurotransmitter release from viable purely cholinergic Torpedo synaptosomes

Viable synaptosomes from the electric organ of Torpedo have been prepared and partially purified. The synaptosomes contain about 100 fold more acetylcholine (Ach) than do mammalian synaptosomes, synaptic vesicles and mitochondria. The Torpedo synaptosomes release Ach by K depolarization in the presence of Ca ions, and manifest an ionophore-mediated Ca-dependent Ach release. These results demonstrate that the synaptosomes contain the neurosecretion apparatus in a functional viable state. Since this preparation uniquely contains only one neurotransmission system (cholinergic), it is most suitable for structural and functional investigations of neuro transmission.     

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.     

Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH(3) cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.     

Arachidonic acid release from K+-evoked depolarization of brain synaptosomes

Rat brain synaptosomes prelabeled with [¹⁴C]arachidonate in their phospholipids were superfused with well oxygenated Krebs-Ringer-bicarbonate solution containing 0.2% BSA and subsequently depolarized by elevating the K⁺ concentration in the superfusion medium from 5 to 55 mM. The efflux of labeled arachidonate at steady state was 0.19% (n = 12) of total radioactivity per min. In the presence of 2.5 mM Ca²⁺, high K⁺ (55 mM) in the medium elicited an increase in arachidonate efflux which amounted to 121.4% (n = 6) of control. Both Ca²⁺ and BSA were required for the stimulated efflux of arachidonate during K⁺-depolarization. Under the same condition, K⁺-stimulation also evoked the release of [³H]norepinephrine which was preloaded into the synaptosomes prior to superfusion. EGTA abolished the stimulated release of both arachidonate and norepinephrine during K⁺-depolarization. These results, together with the loss of labeled arachidonic acid from phospholipids (Majewska and Sun, 1982), indicate that deacylation of membrane lipids is involved in synaptic functions.     

Homeostatic regulation of glutamate release in response to depolarization

Proper nervous system function requires a balance between excitation and inhibition. Systems of homeostasis may have evolved in neurons to help maintain or restore balance between excitation and inhibition, presumably because excessive excitation can cause dysfunction and cell death. This article reviews evidence for homeostatic mechanisms within the hippocampus that lead to differential regulation of glutamate and gamma-aminobutyric acid release in response to conditions of excess depolarization. We recently found differential effects on glutamate release at the level of action potential coupling to transmitter release, vesicular release probability, and vesicle availability. Such differential regulation may help to prevent excitotoxicity and runaway excitation.     

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.     

Action of neurotropic substances on gamma-aminobutyric acid release from brain synaptosomes during K+ depolarization

The effects of aminazine (0.25 mM), phthoracyzine (0.5 mM), trifluperidole (0.5 mM) and imipramine (0.5 mM) on GABA release from rat brain synaptosomes depolarized with K+ (50 mM) were investigated. Incubation of synaptosomes with aminazine led to a 2-fold and that with phthoracyzine, trifluperidole and imipramine to a 1.5-fold increase in GABA release from synaptosomes as compared with its basic level. The raising of K+ in the incubation medium to 50 mM brought about a 2-fold augmentation of GABA release. Exposure of synaptosomes to drugs and a higher K+ concentration at a time did not change GABA release as compared to its basic level. Introduction into the incubation medium of the Ga-ionophore A23187 together with 50 mM K+ and trifluperidole or with K+ and imipramine led to the same increase in GABA release from synaptosomes as that produced by the psychotropic drugs as regards native synaptosomes. It is assumed that the lack of the influence of the psychotropic drugs under study of GABA release from synaptosomes depolarized with K+ is caused by blockade of synaptic membrane conductibility for Ca2+.     

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."     

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.     

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.     

Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax

The binding properties of the purified acetylcholine receptor from $\text{Torpedo}\text{californica}$ were investigated. One type of binding was observed for acetylcholine (K_D = 2.3 μM), dimethyl tubocurarine (K_D = 6.2 μM), and decamethonium (K_D = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature.     

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization,veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [³H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [³H]acetylcholine release in a concentration-dependent manner (5–10 mM); a high concentration (10 mM) also elevated [³H]choline efflux. However, the 35 mM K⁺ induced release of [³H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K⁺ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 M). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K⁺ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

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.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Binding of phencyclidine to the detergent solubilized acetylcholine receptor from Torpedo marmorata

The binding of phencyclidine to the acetylcholine receptor from Torpedo marmorata electroplaque was measured following solubilization of the receptor in sodium cholate followed by the exchange of cholate for Tween 80. In both the membrane-bound and solubilized AChR, the addition of cholinergic agonists simultaneously with the addition of PCP results in a 100 to 1000 fold increase in the PCP association rate and a 5 to 10 fold increase in the dissociation rate as compared to the unliganded AChR or AChR equilibrated with agonist prior to PCP addition. In addition, the number of binding sites and the pharmacological properties of the binding are not markedly changed in the soluble receptor. These results suggest that the acetylcholine receptor can undergo similar conformational transitions in the membrane-bound and the Tween 80 solubilized form and that phencyclidine can monitor these transitions in both cases.