Biomedical subjects
Publications and source records attributed to B Lesbats.
A protein (the mediatophore) purified from Torpedo electric organ nerve terminals was identified by its ability to translocate acetylcholine upon calcium action. The recent large scale production of this protein led us to prepare a polyclonal antibody that was used to study the localization of the protein.
Mediatophore is a protein purified from the nerve terminal membranes of Torpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for the Torpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The activity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.
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 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.
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.
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.
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.
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.
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.
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.
A lyophilized presynaptic membrane powder prepared from Torpedo electric organ synaptosomes was incorporated into liposomes. These proteoliposomes had a large internal volume. The P and E faces of their membrane showed particles which were comparable to the presynaptic membrane ones. The synaptosomal ecto-esterase activity was also incorporated. A large amount of acetylcholine could be entrapped in the proteoliposome which became permeable to acetylcholine in the presence of calcium. Acetylcholine was released in preference to choline. The calcium-induced acetylcholine release depended on the incorporation of a presynaptic membrane constituent. Proteoliposomes prepared from postsynaptic membrane powders gave a much slower acetylcholine efflux. The protein pattern of presynaptic and postsynaptic membrane proteoliposomes were compared.
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.
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.
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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.
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.
Cholinergic synaptosomes were depolarized with KCl or treated with a venom extracted from the annelid Glycera convoluta. This venom was shown to increase considerably the frequency of the miniature endplate potentials at neuromuscular junctions. The synaptosomes were frozen and fractured in the absence of any fixative or cryoprotectant. Synaptic activity decreased the number of small (6 to 8 nm) particles in the P faces of the presynatic membrane, while the large particles (above 8 nm) increased on both P and E faces. It is suggested that these modifications are related to ionic flux or more directly to the release of transmitter.