Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

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.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Cetiedil, a Drug That Inhibits Acetylcholine Release in Torpedo Electric Organ

The effects of cetiedil, a vasodilatator substance with reported anticholinergic properties, were examined on cholinergic presynaptic functions at the nerve electroplaque junction of Torpedo marmorata using either synaptosomes or slices of intact tissue. Cetiedil abolished the calcium-dependent release of acetylcholine (ACh) triggered by depolarization or by addition of A23187 ionophore, a finding localizing the site of action downstream from the calcium entry step. In addition, a direct effect on the release process itself was indicated by the observation that cetiedil blocks the release of ACh mediated by a recently isolated presynaptic membrane protein, the mediatophore, reconstituted into ACh-containing proteoliposomes. In all three preparations, ACh release was inhibited by cetiedil with a Ki of 5-8 microM. Under the conditions used in these release experiments, the synthesis of ACh and its compartmentation within the nerve terminals were not modified. However, the drug was able to reduce high-affinity choline uptake and vesicular ACh incorporation when it was given together with the radioactive precursor, a result showing that cetiedil has a broad inhibitory action on cholinergic uptake processes.     

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

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

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

Presynaptic k-Opioid and Muscarinic Receptors Inhibit the Calcium-Dependent Component of Evoked Glutamate Release from Striatal Synaptosomes

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.     

Presynaptic muscarinic receptor activation enhances striatal dopamine release evoked by depolarization but not that induced by non-depolarizing stimuli

The release of [³H]dopamine stimulated by depolarization with 15 mM KCl of superfused rat striatal synaptosomes was potentiated by acetylcholine through the activation of presynaptic muscarinic receptors. In contrast, acetylcholine did not potentiate the release of [³H]dopamine elicited by d-amphetamine nor that caused by the calcium ionophore A23187. The dopamine carrier blocker nomifensine prevented the releasing action of amphetamine but not that of acetylcholine. The results suggest that the activation of muscarinic receptors on dopamine terminals in the rat corpus striatum selectively affects the calcium-dependent depolarization-induced release of the [³H]catecholamine. Moreover, the [³H]dopamine release caused by acetylcholine seems to occur independently of the membrane dopamine carrier.     

Phosphatidic acid metabolism,calcium ions and transmitter release from electrically stimulated synaptosomes

Synaptosomes isolated from guinea pig brain cortex were stimulated electrically in a medium containing [32P]-orthophosphate. The electrical stimulation caused increased labelling of phosphatidic acid in a synaptic vesicle fraction prepared by osmotic shock of the incubated synaptosomes. Electrical stimulation also provokes transmitter release from the synaptosomes. Both increased phosphatidate labelling and transmitter release required calcium ions in the medium. The effects are discussed in relation to earlier work with acetylcholine and the possible involvement of membrane phosphatidic acid in transmitter release by exocytosis.     

Leptinotarsin-D, a neurotoxic protein, evokes neurotransmitter release from, and calcium flux into, isolated electric organ nerve terminals

Previous work has demonstrated that the neurotoxin leptinotarsin elicits release of neurotransmitter from mammalian nerve terminals, and it has been suggested that the toxin may act either as a direct agonist of voltage-sensitive calcium channels in these terminals (Crosland et al., 1984) or as a calcium ionophore (Madeddu et al., 1985a,b). Preliminary studies (Yeager et al., 1987) demonstrated that leptinotarsin also evokes transmitter release from isolated elasmobranch electric organ nerve terminals. We now report further investigations of the effects of leptinotarsin in this system. The action of the toxin is saturable, releasing about the same small fraction of total transmitter as that released by depolarization. An upper limit for the concentration for half maximal release is estimated to be 4 nM. Leptinotarsin-evoked transmitter release exhibits behavior very similar to depolarization-evoked release with respect to dependence on Ca2+, Ba2+, and Sr2+ and blockade by Co2+, Cd2+, and trifluoperazine. Leptinotarsin also promotes the uptake of calcium into synaptosomes to a degree similar to that caused by depolarization by K+. The binding of leptinotarsin to nerve terminals is probably Ca2+ dependent and receptor mediated. Taken together with the behavior of leptinotarsin-evoked release in other preparations, these results are consistent with the hypothesis that this toxin acts by opening a presynaptic calcium channel. However, the possibility that leptinotarsin is a calcium ionophore cannot be excluded.     

Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis

Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine-radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium-dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins.     

How does calcium trigger neurotransmitter release?

Recent work has established that different geometric arrangements of calcium channels are found at different presynaptic terminals, leading to a wide spectrum of calcium signals for triggering neurotransmitter release. These calcium signals are apparently transduced by synaptotagmins - calcium-binding proteins found in synaptic vesicles. New biochemical results indicate that all synaptotagmins undergo calcium-dependent interactions with membrane lipids and a number of other presynaptic proteins, but which of these interactions is responsible for calcium-triggered transmitter release remains unclear.     

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

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

12-Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings

Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium-dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12-lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5- or 15-lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-10 microM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12-lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.     

A Role for Transglutaminase in Neurotransmitter Release by Rat Brain Synaptosomes

Rat brain synaptosomes exhibit calcium-dependent transglutaminase activity. This activity, measured in detergent-treated or sonicated preparations, was six- to sevenfold lower than that in the liver. The synaptosomal transglutaminase was inhibited by various amines and alpha-difluoromethylornithine, compounds known to inhibit activity of this enzyme in other tissues. The inhibitors of transglutaminase induced release of catecholamines, but not of gamma-aminobutyric acid, from synaptosomes both under basal and K+-stimulated conditions. The concentrations of the agents that caused stimulation of catecholamine release were approximately the same as those that inhibited the activity of transglutaminase. Stimulation of release was largely reduced by the withdrawal of calcium from the incubation medium. Inhibitors of transglutaminase had little effect either on the uptakes of neurotransmitters or the amounts of deaminated products of catecholamine degradation released into the medium. It is suggested that a synaptosomal transglutaminase is involved in suppressing vesicular release of catecholamines by resting (nondepolarized) neurons and that this action may also be a part of negative feedback control which prevents excessive transmitter release at the synapse during increased neuronal activity.     

Large-Scale Purification of Torpedo Electric Organ Synaptosomes

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

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.     

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

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

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.