Bernard Lesbats

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

Abstract: 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.

Application to Mammalian Tissues of the Chemiluminescent Method for Detecting Acetylcholine

Abstract: It is now possible to extend to mammalian tissues the chemilumi‐nescent 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.

A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore

Upon SDS PAGE of isolated mediatophore, an acetylcholine‐translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue‐long peptide completely homologous with a sequence of the proton‐translocating proteolipid from bovine chromaffin granules. A 51‐mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in λZap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

Retrograde Inhibition of Transmitter Release by ATP

Abstract: After labelling ACh tissue stores in Torpedo electric organ prisms with radioactive acetate, the release of ACh and ATP triggered by electrical stimulation or KCI depolarization was measured in the same perfusate samples. The luciferin‐luciferase reaction for ATP was first counted, then the radioactive content of the sample determined. Further evidence showing that ATP release resulted from postsynaptic transmitter action was that carbachol could induce the release of ATP. A dose‐response curve was obtained. Curare or α‐bungarotoxin block the release of ATP elicited by carbachol. When triggered by KCI depolarization the increased efflux of ACh and ATP returned to low levels in spite of the maintained depolarization. After two successive KCI depolarizations, it was possible to dissociate the release of both substances. The efflux of ATP was exhausted while ACh release was maintained. If the second KCI depolarization was delayed ATP release recovered, but the release kinetics of ACh and ATP were sustained. The exhaustion of endogenous ATP release or the action of exogenous ATP had little or no effect on the release of ACh triggered by KCI depolarization. On the contrary, the release of ACh induced by electrical stimulation was sensitive to the action of adenine nucleotides, and a quantitative estimation of the inhibition of ACh release by ATP and adenosine could be made. At the onset of stimulation ATP release predominated, being gradually replaced by adenosine, which can be reuptaken. This would terminate the inhibitory action of the nucleotide. Carbachol inhibits evoked ACh release, while the effect of α‐bungarotoxin was to increase spontaneous ACh release. These effects could be respectively mediated by an increased or a reduced release of ATP resulting from the postsynaptic action of ACh agonists or antagonists. However, a direct presynaptic effect of these substances is not excluded. It seems possible that the action of ATP on ACh release can be explained through its inhibition of the depolarization‐evoked Ca2+ entry.

RELATED CHANGES IN AMOUNTS OF ACh AND ATP IN RESTING AND ACTIVE TORPEDO NERVE ELECTROPLAQUE SYNAPSES

Abstract— Closely related changes in the levels of acetylcholine (ACh) and adenosine triphosphate (ATP) in the electric organ of Torpedo exist during rest and synaptic activity. The present work clarifies these relations by showing:

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium‐Dependent Acetylcholine Release from Proteoliposomes

Abstract: 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.

Redistribution of intramembrane particles related to acetylcholine release by cholinergic synaptosomes

Acetylcholine release was measured on suspensions of pure cholinergic synaptosomes, isolated from torpedo electric organ. Transmitter release was triggered by two different methods: KCl depolarization, or action of a venom extracted from a polychaete annelid Glycera convoluta . This venom was known to increase considerably the miniature endplate potential frequency at neuromuscular junctions. Ultrarapid freezing of synaptosomes in suspension in the absence of fixation, followed by freeze fracture, permitted us to show: (1) That the venom does not trigger the appearance of endo-exocytotic pits in the presynaptic membrane, in contrast to KCl depolarization. (2) That both KCl depolarization and venom action lead to a decrease in the number of small P-face intramembrane particles and to an increase in the number of medium-sized E-face particles. In addition, the venom increased the number of medium-sized P-face particles. The redistribution of the intramembrane particles is discussed in relation to the release of transmitter which has been measured in parallel.

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15‐kDa subunit of mediatophore and a 14‐kDa membrane protein that has a wide non‐neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15‐kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14‐kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14‐kDa antigen raising the question of a possible interaction of mediatophore with the 14‐kDa protein originating from the Schwann cell.

Is the acetylcholine releasing protein mediatophore present in rat brain

Mediatophore is a protein purified from the nerve terminal membranes ofTorpedo 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 theTorpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The acitivity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.

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

Summary— 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.