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Biomedical subjects

M Israël

Publications and source records attributed to M Israël.

At least 73 records · Page 4Linked to original sources
PubMed1987

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.

Acetylcholine↗
PubMed1987

[Cholinergic chemical transmission. Mechanisms of control].

The isolation of fractions of purified synaptic constituents has permitted to localize the enzymes involved in cholinergic mechanisms and to evaluate the acetylcholine concentration of vesicular stores and of the cytoplasmic pool where transmitter is synthetized. The isolation of cholinergic synaptosomes allowed study the mechanisms and regulations controlling the release process. The depletion of cytoplasmic acetylcholine elicited by stimulation is discussed in parallel to the role of synaptic vesicles. Presynaptic membrane proteins seem to be directly involved in the release of the transmitter.

Acetylcholine↗
PubMed1986

Purification of a presynaptic membrane protein that mediates a calcium-dependent translocation of acetylcholine.

A protein, which we call "mediatophore," that mediates calcium-dependent release of acetylcholine from proteoliposomes has been purified from the presynaptic plasma membrane. About 250 micrograms of this material was obtained from 500 g of Torpedo marmorata electric organ. Precipitation of the protein and subsequent removal of associated lipids inactivated the protein, which then became water soluble; this permitted evaluation of its Stokes radius (52 A) and its sedimentation coefficient (9.8 +/- 0.75 S) and, hence, an approximate molecular mass of 210 +/- 16 kDa could be determined. PAGE analysis showed that the protein is made of 17-kDa subunits, not linked by disulfide bonds. When this material was observed by electron microscopy after negative staining, the apparently pentameric structures had an average diameter of about 7 nm.

Acetylcholine↗
PubMed1986

Solubilization and partial purification of a presynaptic membrane protein ensuring calcium-dependent acetylcholine release from proteoliposomes.

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.

Acetylcholine↗
PubMed1985

Acetylcholine release in the cat caudate nucleus measured with the choline oxidase method.

A chemiluminescent assay for the estimation of acetylcholine (ACh) was used to measure ACh release in caudate nuclei (CN) of halothane-anaesthetized cats implanted with push-pull cannulae. The validity of the entire experimental approach used was shown by the fact that ACh release was calcium-dependent and was increased by depolarizing agents (potassium ions, veratridine) as well as by atropine. The effects of GABA (10(-5) M, 30 min) unilateral application into the ventralis medialis and ventralis lateralis thalamic nuclei on ACh release in both CN were then examined. This treatment, known to increase DA release bilaterally, decreased ACh release in both CN. These data further reveal the role of thalamic nuclei in the bilateral regulation of the activity of neurons identified within the basal ganglia and are discussed in the light of the well-known inhibitory influence of nigrostriatal DA neurons on striatal cholinergic neurons.

Acetylcholine↗
PubMed1985

Large-scale purification of Torpedo electric organ synaptosomes.

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.

Acetylcholine↗
PubMed1985

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↗
PubMed1984

Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation.

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces. This phenomenon resembles the intramembrane particle rearrangements found in stimulated synaptosomes. No visible structures capable of releasing acetylcholine as a result of the calcium influx were found inside the proteoliposomes. This supports the view that the release of free cytosolic acetylcholine from stimulated nerve terminals can be directly attributed to presynaptic membrane proteins. These proteins were extracted in a functional form from the synaptosomal membrane.

Acetylcholine↗
PubMed1982

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein.

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase. Gel electrophoresis permitted the identification of a major protein component of the presynaptic membrane fraction which had a molecular weight of 67,000. This protein was not found in postsynaptic membrane or synaptic vesicle fractions. Thus it appeared to be specific to the nerve terminal plasma membrane.

Acetylcholine↗
PubMed1982

Application to mammalian tissues of the chemiluminescent method for detecting acetylcholine.

It is now possible to extend to mammalian tissues the chemiluminescent acetylcholine assay. Mammalian tissue extracts must be treated with oxidants (which is not necessary for electric organ extracts). The assay can then be performed as previously described (acetylcholinesterase hydrolyses acetylcholine; choline oxidase converts choline to betaine and H2O2, which gives off light in the presence of luminol and peroxidase). It is also shown that release experiments can be performed on mammalian tissue slices (mouse caudate nucleus) after the slice is washed in oxygenated saline solutions.

Acetylcholine↗
PubMed1982

Rearrangement of intramembrane particles as a possible mechanism for the release of acetylcholine.

1. A chemiluminescent procedure for measuring acetylcholine (ACh) has recently been described. The procedure is based on the hydrolysis of ACh by acetylcholinesterase and on the oxidation of choline to betaine and H2O2 by choline oxidase. The H2O2 generated reacts with luminol in presence of peroxidase to produce a light emission. This method is sensitive in the pmol/ml range. 2. On isolated synaptosomes from electric organ, it is possible to obtain an estimate of the cytoplasmic ACh compartment by measuring the light emission after a single freezing and thawing cycle. The vesicular pool which resists several freezing and thawing cycles is then estimated by opening the compartment with a detergent. Increasing the intensity of stimulation of synaptosomes with different agents depletes the ACh content down to the vesicular pool. 3. The release of ACh is not associated with any change in the number of synaptic vesicles as seen in cryofractured synaptosomes. The only ultrastructural change detected common to all stimulations was a decreased density of P face intramembrane particles smaller than 11 nm and an increased density of E face 8 to 18 nm particles. The very significant particle changes were more intense for the conditions releasing more ACh. It is suggested that these particles are involved in the release of ACh from the cytoplasm. An attempt to directly correlate the release of ACh with intramembrane particle changes is discussed.

Acetylcholine↗
PubMed1982

Partial purification of the Glycera convoluta venom components responsible for its presynaptic effects.

The crude extract of glands appended to the jaws of the polychaete annelid Glycera convoluta induces an important increase in the spontaneous quantal transmitter release on frog and crayfish neuromuscular junctions and on Torpedo nerve-electroplaque junctions. The venom similarly triggers acetylcholine (ACh) release from synaptosomes purified from Torpedo electric organ. At the frog neuromuscular junction, the reproducibility, the reversibility and the dose-dependence of the venom action permit a quantitative evaluation of the effect. The crude venom extract has been fractionated by gel-filtration. The effect on transmitter release has been found in a high molecular weight fraction distinct from those which contain the protease and phospholipase activities.

Acetylcholine↗
PubMed1982

Evidence for a specific protein associated to the plasma membrane of cholinergic synaptosomes.

Synaptosomal plasma membrane fractions were prepared by fractionation of pure Torpedo cholinergic synaptosomes. A 67 000 dalton peptide was shown to be a major component of the presynaptic membrane. It appears specific for this membrane since (1) it copurifies with the synaptosomal plasma membrane; (2) it was not present in similar plasma membranes but prepared from Torpedo electric lobes or electric nerves, and since (3) rabbit antibodies to presynaptic antigens which were mainly directed to this 67 000 dalton peptide band were shown to bind to the nerve terminal network in Torpedo electric organ. A crude fraction of presynaptic plasma membrane prepared from electric organ homogenate could be a convenient material for the purification of this 67 000 dalton peptide.

Animals↗
PubMed1981

Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpedo electric organ synaptosomes.

The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60-70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.

Acetylcholine↗
PubMed1980

[Continuous detection of the release of acetylcholine from the electric organ of Torpedo using a chemiluminescence reaction].

A chemiluminescent procedure to measure acetylcholine was developed and used to detect continuously the release of transmitter from stimulated electric organ slices and synaptosomes. Acetylcholine is hydrolysed by acetylcholinesterase; the choline liberated is oxidized by choline oxidase to betaïne; the H2O2 generated triggers the luminescence of luminol in presence of peroxidase. The extension to other transmitters is possible; several developments are envisaged.

Acetylcholine↗