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Biomedical subjects

M Israël

Publications and source records attributed to M Israël.

At least 19 recordsLinked to original sources
PubMed1996

Cell lines expressing an acetylcholine release mechanism; correction of a release-deficient cell by mediatophore transfection.

Several neuronal and non-neuronal cell lines express a Ca(2+)-dependent mechanism of transmitter release that can be demonstrated after loading the cells with acetylcholine during culture. In contrast, a particular cell line, the neuroblastoma N18TG-2, was found to be deficient for release. We transfected N18TG-2 cells with a plasmid encoding Torpedo mediatophore, a protein able to translocate acetylcholine in response to calcium. The N18TG-2 cells expressed the Torpedo protein which reached their plasma membrane. At the same time, these cells acquired a Ca(2+)-dependent quantal release mechanism similar to the one naturally expressed by other cell lines. Hence, the presence of mediatophore in the plasma membrane seems essential for quantal release.

Acetylcholine↗
PubMed1996

Activation and desensitisation of acetylcholine release by zinc at Torpedo nerve terminals.

Treatment with 100 or 250 microM ZnCl2 irreversibly blocked neurotransmission in the Torpedo electric organ by inhibiting acetylcholine (ACh) release. In Zn2+-treated tissue, release failure did not result from impairment of Ca2+ entry since stimulation still provoked an accumulation of Ca2+. Also pretreatment of isolated synaptosomes with Zn2+ inhibited to the same extent the release elicited by KCl-evoked depolarisation and the release elicited by using the Ca2+ ionophore A23187. On the other hand, after application of A23187, Zn2+ by itself efficiently triggered ACh release from synaptosomes. This dual effect of Zn2+ was also observed to occur in proteoliposomes equipped with mediatophore (a protein of the presynaptic membrane characterised by its capability to support Ca2+-dependent transmitter release). Hence, Zn2+ mimicked two fundamental actions of Ca2+ on nerve terminals, which are: (1) the immediate activation of release, and (2) a more slowly developing desensitisation of release. Zn2+ was more powerful than Ca2+ for both actions. It is concluded that the dual action of Zn2+ on the mediatophore protein accounts at least in part for its complex effects on neurotransmission.

Acetylcholine↗
PubMed1996

A unifying hypothesis for acetylcholine release.

Mediatophore is the only nerve terminal membrane protein known to translocate acetylcholine upon calcium action. It is localized at the active zone. In this review we attempted to describe its role in relation to the vesicular and membrane protein complexes that are formed at the active zone. The model pictures a possible set of sequential steps that lead to exocytosis. The smallest quantal events are attributed to mediatophore opening momentarily, while synaptic vesicles synchronize release by controlling the calcium microdomain. A clear distinction is made between sub-quantal ACh release preserved after Botulinum toxin action, and exocytosis of vesicular contents. A cybernetic model for release and exocytosis related to protein interactions is presented for future works.

Acetylcholine↗
PubMed1996

Evoked acetylcholine release expressed in neuroblastoma cells by transfection of mediatophore cDNA.

Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.

Acetylcholine↗
PubMed1995

Mediatophore and other presynaptic proteins. A cybernetic linking at the active zone.

In rapidly transmitting synapses, the mediatophore, a protein located in the presynaptic membrane, seems to play a key role in the last step of transmitter release. Reconstituted either in proteoliposomes or in Xenopus oocytes, or transfected in particular cell lines, the mediatophore is able to release acetylcholine with characteristics which meet several typical features of transmitter release in natural synapses. Good correspondence between the two conditions was found for: i) the dependency of release upon calcium concentration; ii) the desensitisation of release by persistence of internal calcium; iii) the effect of several drugs; iv) the fleeting formation of a population of large intramembrane particles during the precise time of release; and v) the pulsatile or quantal nature of transmitter release. All these features therefore could well be ascribed to intrinsic properties of the mediatophore molecule. How is the mediatophore integrated in the whole presynaptic apparatus? To what extent is its function regulated by the other proteins of the active zone? These questions are far from being solved. We want nevertheless to propose here a general view in which characteristic presynaptic functions such as transmitter release, calcium entry, sequestration and extrusion, regulation of short- and long-term changes in release efficiency, are supported by an ordered succession of molecular events involving the proteins of the active zone. It will be seen that some proteins compete for a common binding site. It is thus expected that they will occupy this site in a regulated succession, according to simple cybernetic rules.

Animals↗
PubMed1995

Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter.

The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription-PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.

Animals↗
PubMed1994

Acetylcholine accumulation and release by hybrid NG108-15, glioma and neuroblastoma cells--role of a 16kDa membrane protein in release.

A procedure is described to fill up cells in culture with ACh and study its calcium dependent release, by-passing the synthesis steps. Whether differentiated or not with dbc-AMP, the NG108-15 cells efficiently released ACh when stimulated with calcium and ionophore A23187. The release was also studied in the parent C6-BU-1 and N18TG2 cells. It was found that C6-BU-1 released ACh much better that N18TG2 in spite of their glial origin. The internalization by NG108-15 cells of an antisense oligonucleotide probe hybridizing the 16 kDa proteolipid messenger common to mediatophore and to the V-ATPase reduced ACh release indicated a role of this proteolipid in ACh translocation. This characteristic protein was found in the membrane extract of NG108-15 cells and also in the C6-BU-1 cells, but its amount was strongly reduced in the N18TG2 cell line and in the NG108-15 cells having internalized the antisense probe.

Acetylcholine↗
PubMed1994

In vitro expression of the 15 kDa subunit of the mediatophore and functional reconstitution of acetylcholine release.

The mediatophore is a presynaptic oligomeric protein purified from the presynaptic plasma membrane of Torpedo synaptosomes on the basis of its ability to mediate a calcium-dependent acetylcholine release when solubilized and reconstituted into proteoliposomes. We investigated the ACh translocating activity of the 15 kDa proteolipid subunit of the mediatophore when expressed in Xenopus oocytes and reconstituted into proteoliposomes loaded with ACh. 1. A calcium-dependent ACh translocation was observed when oocytes were injected with polyadenylated mRNAs extracted from the electric lobe of the Torpedo brain or with an in vitro transcribed RNA encoding the 15 kDa subunit. 2. No release response was obtained when oocytes were non-injected or injected with Torpedo liver mRNAs. 3. This ACh translocation mechanism showed calcium-dependent activation and desensitisation and was inhibited by cetiedil, sharing these properties with the release of ACh observed at the synapse. 4. The ACh translocating activity of an N terminal deleted mediatophore 15 kDa subunit was strongly reduced and the deleted proteolipid appeared less sensitive to the action of cetiedil (alpha-cyclohexyl-alpha-(3-thienyl)-acetate of perhydroazepinyl-alpha-ethyl citrate monohydrate). 5. A significant ACh release response was observed when the 15 kDa proteolipid of the H(+)-ATPase from bovine chromaffin granules was tested. 6. These results show that this ACh translocating activity could be induced in the oocyte membranes by the expression of the 15 kDa subunit alone.

Acetylcholine↗
PubMed1994

Spontaneous release of acetylcholine from Torpedo synaptosomes: effect of cetiedil and its analogue MR 16728.

The effects of cetiedil and its analogue MR 16728 were examined on spontaneous acetylcholine release measured with a chemiluminescent assay using choline oxidase in a synaptosomal suspension obtained from Torpedo marmorata electric organ. Evoked acetylcholine release is inhibited by cetiedil, whereas this drug enhances spontaneous extracellular Ca(2+)-independent acetylcholine release (up to 340%). This effect was examined as a function of cetiedil concentration and incubation time. On the other hand, the analogue MR 16728, which enhances A23187-evoked acetylcholine release, also enhances spontaneous Ca(2+)-independent acetylcholine release. Cetiedil and MR 16728 effects on spontaneous acetylcholine release were also examined in the presence of Ca2+. Addition of Ca2+ enhanced spontaneous acetylcholine release by 75%, and cetiedil and MR 16728 stimulation was maintained but with different levels of enhancement. Thus, these results show that the processes responsible for evoked and spontaneous acetylcholine release are sensitive but in different ways to drugs of the cetiedil family.

Acetamides↗
PubMed1993

Antisense probes against mediatophore block transmitter release in oocytes primed with neuronal mRNAs.

Antisense oligodesoxynucleotides were used to determine whether the mediatophore proteolipid is necessary for the Ca(2+)-dependent release of the neurotransmitter acetylcholine. Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain an homogeneous population of cholinergic neurons homologous to motoneurons. Addition of antisense probes hybridizing to the mediatophore 15 kDa subunit inhibited the expression of both the mediatophore proteolipid in oocyte membranes and the Ca(2+)-dependent acetylcholine release. Expression of other neuronal functions such as synthesis of [14C]acetylcholine from [14C]acetate was not inhibited. Another antisense probe specific for the sequence of a related proteolipid cDNA (the 15 kDa subunit of the chromaffin granule protonophore) was used as a control. It did not hybridize with the Torpedo mediatophore mRNA and, injected in addition to electric lobe mRNAs, it did not inhibit either mediatophore expression or acetylcholine release. We showed in addition that the mRNA primed oocytes did not contain a vesicular pool of acetylcholine. It was concluded (i) that the mediatophore proteolipid is essential for Ca(2+)-dependent acetylcholine release and (ii) that the cytosolic pool of neurotransmitter seems to be preferentially used in this system.

Acetylcholine↗
PubMed1993

The effect of MR16728, a cetiedil analogue, on acetylcholine release in Torpedo synaptosomes.

MR16728, a cetiedil analogue, enhanced acetylcholine (ACh) release (up to 145% of control) from Torpedo synaptosomes when the release was triggered by a Ca2+ ionophore, A23187 or ionomycin, in the presence of 4 mM Ca2+ in the release medium, but inhibited ACh release induced by KCl depolarization of the presynaptic membrane. MR16728 also inhibited Ca(2+)-ATPase activity measured in purified synaptosomal presynaptic membranes. We studied the stimulation by MR16728 as a function of its concentration; the half-maximal effect was reached at the concentration of 13.5 microM. Moreover the stimulation was more pronounced (up to 300%) when a low concentration of Ca2+ (in the 10-micromolar range) was added to the medium. The enhancement of ACh release was also observed in proteoliposomes having incorporated mediatophore, a presynaptic membrane protein, able to release ACh in a Ca(2+)-dependent manner. Thus, mediatophore is a potential presynaptic target for MR16728.

Acetamides↗
PubMed1993

Glutamate and acetylcholine release from cholinergic nerve terminals, a calcium control of the specificity of the release mechanism.

We describe, in the present work, a continuous procedure for measuring the release of glutamate, and have applied it to analyze the co-release of glutamate and acetylcholine from cholinergic nerve terminals of electric organ synaptosomes. The two substances were measured in similar conditions using the two continuous chemiluminescent assays. The protein "Mediatophore" reconstituted in proteoliposomes was also able to translocate both transmitters with a preference for glutamate when the external calcium was increased between 3 and 10 mM. However, at lower calcium concentrations a clear preference for acetylcholine was found. For a regulated calcium entry as obtained by electrical stimulation of the nerve the release mechanism was fully specific for acetylcholine. Since the specificity of mediatophore depends on the local calcium amounts, the possible role of mediatophore at acetylcholine or glutamate specific synapses may be envisaged and is discussed in relation to the calcium control of specificity.

Acetylcholine↗
PubMed1993

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.

Acetylcholine↗
PubMed1992

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

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.

Acetylcholine↗