N-ethylmaleimide-sensitive factor is associated with the nuclear envelope

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.     

Lanthanides induce neurotransmitter release from the vesicular pool in rat brain synaptosomes

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.     

Cycling of actin assembly in synaptosomes and neurotransmitter release

We have investigated the regulation of actin assembly in whole mouse brain synaptosomes and how that regulation modulates neurotransmitter release. During a 30 s depolarization with high K+, filamentous actin (F-actin) levels, monitored by staining with rhodamine phalloidin, increase dramatically (up to 300% in 3 s), decrease, and increase once again. This F-actin cycling is regulated by pathways both dependent and independent of Ca2+ influx and is markedly affected by exposing synaptosomes to Li+, tetrodotoxin, and diacylglycerol. Measurement of [3H]norepinephrine release from synaptosomes containing entrapped agents that modulate actin assembly (DNAase I or phalloidin) indicates that actin depolymerization is necessary for normal release and that repolymerization limits release.     

l-Aspartate as an amino acid neurotransmitter: mechanisms of the depolarization-induced release from cerebrocortical synaptosomes

The role of l -aspartate as a classical neurotransmitter of the CNS has been a matter of great debate. In this study, we have characterized the main mechanisms of its depolarization-induced release from rat purified cerebrocortical synaptosomes in superfusion and compared them with those of the well-known excitatory neurotransmitter l -glutamate. High KCl and 4-aminopyridine were used as depolarizing agents. At 15 mM KCl, the overflows of both transmitters were almost completely dependent on external Ca²⁺. At 35 and 50 mM KCl, the overflows of l -aspartate, but not those of l -glutamate, became sensitive to dl -threo-β-benzyloxyaspartic acid ( dl -TBOA), an excitatory amino acid transporter inhibitor. In the presence of dl -TBOA, the 50 mM KCl-evoked release of l -aspartate was still largely external Ca²⁺-dependent. The dl -TBOA insensitive, external Ca²⁺-independent component of the 50 mM KCl-evoked overflows of l -aspartate and l -glutamate was significantly decreased by the mitochondrial Na⁺/Ca²⁺ exchanger blocker CGP 37157. The Ca²⁺-dependent, KCl-evoked overflows of l -aspartate and l -glutamate were diminished by botulinum neurotoxin C, although to a significantly different extent. The 4-aminopyridine-induced l -aspartate and l -glutamate release was completely external Ca²⁺-dependent and never affected by dl -TBOA. Superimposable results have been obtained by pre-labeling synaptosomes with [³H] d -aspartate and [³H] l -glutamate. Therefore, our data showing that l -aspartate is released from nerve terminals by calcium-dependent, exocytotic mechanisms support the neurotransmitter role of this amino acid.     

Influence of calcium ionophore A23187 on neurotransmitter release in rat brain synaptosomes

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.     

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.     

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin.

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by microM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.     

Induced enzyme release from synaptosomes by halothane

GABA-transaminase has been found to be released from rat brain synaptosomes by halothane in a dose-related manner. The releases of both GABA-transaminase and succinic semialdehyde dehydrogenase were increased with time. The release of other enzymes (creatine kinase, glutamate decarboxylase, aspartate transaminase, lactate dehydrogenase, and malate dehydrogenase) was less in magnitude and not related to the duration of incubation. Such observations suggested a specific event in the halothane-induced release of GABA-catabolizing enzymes. A suggestion linking mode of anesthetic action to a mitochondrial effect of volatile anesthetics was made.     

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.     

Presynaptic modulation of amino acid release from synaptosomes

Using synaptosomes prepared from whole rat brain, the spontaneous, calcium-independent, and calcium-dependent release of glutamate and GABA was assessed. Time intervals of 1–30 seconds were studied. Spontaneous release of glutamate (but not GABA) was elevated by 10 M NMDA or AMPA by thirty seconds. This stimulation was partially calcium-dependent. Calcium-dependent release induced by 30 mM KCl was biphasic, confirming previous findings. This release was stimulated at all time periods by the presence of 10 M NMDA or AMPA in an antagonist-sensitive manner. These data suggest that glutamate and GABA are released from vesicular stores in rat synaptosomes and that some of this release is modulated by presynaptic glutamate receptors.     

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.     

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.     

Kinetics ofd-aspartic acid release from rat cortical synaptosomes

A kinetic study has been made of the release ofd-aspartate from rat cortical synaptosomes following pre-loading with labelled D-aspartate, and the results compared to a previous study of the release of the acidic amino acids glutamate plus aspartate following pre-loading with labeledl-glutamate. Qualitatively, the results of the two studies are similar. The D-aspartate taken up during the preload period appears to be totally releasable. However, release is greatly increased by depolarizing media. The increased rate of release induced by increasing [K]_o is independent of the [Ca]_o, while veratrine-induced release is inhibited by [Ca]_o. Release is from more than a single compartment, since plots of the log₁₀ of the synaptosomal D-aspartate content (calculated from the label content) as a function of the incubation time are non-linear for all incubation solutions. In the previous study which utilizedl-glutamate pre-loading, the results were consistent with either a model consisting of two passive compartments (that is, synaptosomal content T as a function of time is given by Ae^–Kat+Be^–Kbt, in which A and B are compartment sizes, Ka and Kb are exchange constants, and t is incubation time) or a model consisting of one passive compartment (Ae^–Kat) and one saturated carrier compartment (T-Kbt, in which T=total content at zero time and Kb=maximal velocity). The present results withd-aspartate also give excellent fits to these models. However, there are some quantitative differences in the estimates of the compartment sizes and exchange constants, which are obtained by optimizing the fit of the data to the equation for each model. Although most of these quantitative differences appear to be minor, one difference between the two studies is of potential significance in interpretation of the results. In the glutamate study, all depolarizing media were found to reduce the exchange constant for the carrier mechanism, while in the present study, depolarizing media were found to increase the exchange constant, with the exception of veratrine-containing medium without calcium.     

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.     

Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events

Activation of the phagocytic cell superoxide-generating NADPH oxidase requires interaction of cytosolic and membrane-associated components. With most stimuli activation of the oxidase is accompanied by multisite phosphorylation of the 47-kDa cytosolic oxidase factor (p47) which translocates from cytosol to membranes. Native p47 is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events. Phosphorylation of p47 was studied by immunoprecipitation from neutrophil cytosol and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography. In the resting cell p47 was not phosphorylated. In the cytosol of phorbol myristate acetate-activated neutrophils eight distinct p47 phosphoproteins were present. The membrane fraction from these activated cells contained a family of p47 phosphoproteins of electrophoretic mobilities identical to those seen in cytosol plus an additional, more acidic p47 phosphoprotein not present in cytosol. Very early after activation (30 s) only the four most acidic p47 phosphoproteins were present in the membrane fraction. Only at later times (5-15 min) was the full spectrum of p47 phosphoproteins present in the membrane fraction. In contrast, the full spectrum of p47 phosphoproteins was present in the cytosol over the entire time course we studied. In neutrophils from patients with cytochrome b558-deficient chronic granulomatous disease p47 phosphorylation was incomplete and p47 translocation to membrane did not occur. These studies demonstrated that the cytochrome was essential for formation of the three most acidic p47 phosphoproteins and greatly augmented formation of the fourth most acidic p47 phosphoprotein found in normal neutrophils. The temporal correlation between specific p47 phosphorylation events and p47 translocation to membrane is consistent with a model of oxidase activation in which a series of p47 phosphorylation events which occurs in cytosol precedes and may be required for p47 interaction with membrane.