R. Manaranche

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:

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.

DETERMINATION OF ACh CONCENTRATION IN TORPEDO SYNAPTOSOMES

Abstract— The concentrations of ACh and ATP of Torpedo electric organ synaptosomes were directly measured and found to be respectively 20.0 ± 6.4 mM and 3.1 ± 0.6 mM. The synaptosomal volume was estimated by a classical space marker technique using 14C inulin and tritiated water. After counting the synaptosomes in an haemocytometer (and knowing their volume), a mean diameter of 3.5 p was calculated. The use of these classical techniques was rendered possible because of the homogeneity of the fraction and the large size of Torpedo synaptosomes.

Ultrastructural changes and transmitter release induced by depolarization of cholinergic synaptosomes: A freeze-fracture study of a synaptosomal fraction from torpedo electric organ

A Ca-dependent transmitter release was triggered by K+ depolarization in pure cholinergic synaptosomes isolated from Torpedo electric organ. The synaptosomes were freeze fractured after ultrarapid freezing in the absence of any fixation. A quantitative analysis of freeze-fracture micrographs shows a Ca-dependent increase of the number of pits in the presynaptic membrane upon K+ depolarization. We were able to distinguish two populations of pits after K+ depolarization with or without 4-aminopyridine. This drug reduced the number of the large (20 to 150 nm) pits and increased the number of the small (10 nm) pits while ACh release was unaffected or enhanced. The large pits might represent endoexocytotic events. The small pits, found on the P face without symmetric holes on the E face, could represent either “synaptopores” related to synaptic vesicles or holes left in the P face by specialized particles detached with the E face. These micropits might well be the site of ACh release.

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.

Acetylcholine release from proteoliposomes equipped with synaptosomal membrane constituents

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.

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.

Characterization of a Presynaptic Membrane Protein Ensuring a Calcium‐Dependent Acetylcholine Release

In Torpedo electric organ, acetylcholine (ACh) release was studied at four different levels: intact nerve electroplaque synapses, isolated nerve terminals (synaptosomes), resealed synaptosomal membrane sacs, and proteoliposomes into which presynaptic membrane proteins were incorporated. Electrical stimulation of nerve endings in situ in the whole tissue mobilized the cytoplasmic pool of neurotransmitter, which was released and renewed before any detectable change of the ACh pool contained in synaptic vesicles.”* In response to a calcium influx triggered by various agents, isolated synaptosomes released most of their cytoplasmic ACh without any depletion of the vesicular ACh pool.’ Synaptosomal ghosts refilled with ions and soluble ACh also exhibited a strictly calcium-dependent ACh release.’ It was then possible to go one step further and to incorporate presynaptic membrane constituents into liposomal membranes made of synthetic lecithin, which have entrapped soluble ACh. The proteoliposomes thus obtained released ACh in response to a calcium influx.’ A similar finding was observed using rat brain presynaptic membranes: ACh release from proteoliposomes fulfilled several criteria expected for a physiologically relevant mechanism.’ (1) It was calcium dependent; ACh was released only when calcium entered the proteoliposomes and magnesium could not replace calcium. (2) The kinetics of ACh release from proteoliposomes and synaptosomes were very similar. (3) A presynaptic membrane protein was involved that was not found in plasma membranes of the electroplaques. This integral membrane protein possessed an intracellular domain necessary for the ACh-translocating properties. (4) Finally, ACh was released from proteoliposomes in preference to choline or ATP when these substances were also present inside the liposomes with ACh. Recently, we have used the reconstitution of ACh release in liposomes as a functional test to punfy the membrane protein involved. This protein, which we propose to call mediatophore, was puri6ed according to a three-step procedure: purification of the presynaptic plasma membrane according to a large-scale procedure,8 extraction at alkaline pH, and solubilization in organic solvents. The purified protein was associated to lipids and still active (RG. la). The calcium-dependent ACh release from proteolipsomes was proportional to the amount of mediatophore used for the re-

Intramembrane Particles Changes: A Constant Feature of the Release Mechanism

ABSTRACT The release of acetylcholine measured with a new chemiluminescent method was triggered by KCl in presence or absence of 4 aminopyridine, and after gramicidin or calcium ionophore A 23187 actions. In all conditions studied presently and previously, cryofracture studies of the presynaptic membrane show that the release of acetylcholine is correlated to a decrease of the number of small P face intramembrane particles (≤ 10 nm) and to an increase of larger E face particles (8 to 16 nm). The number of synaptic vesicles remains constant and pits are not systematically found in release conditions able to deplete the cytoplasmic acetylcholine compartment.