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Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

The presumptive choline transporter, CTL1, was initially identified through functional complementation of a triple yeast mutant (ctr ise URA3delta) with deficiencies in both choline transport and choline neosynthesis under selective conditions that cause perturbations in membrane synthesis and growth. After transformation of these yeasts with a heterologous yeast expression library made from Torpedo electric lobe cDNAs, several colonies showed increased growth but only one clone increased the accumulation of external choline. The corresponding full-length cDNA was isolated and encodes a protein with 10 transmembrane domains. Northern analysis of Torpedo mRNA indicates that CTL1 is expressed at high levels in the spinal cord and brain. In Xenopus oocytes, Torpedo CTL1 expression was associated with the appearance of sodium independent high-affinity choline uptake. We propose that CTL1 plays a role in providing choline for membrane synthesis in the nervous system.

We initially isolated CTL1 from the electric lobe of brain through functional complementation of a yeast mutant deficient in choline transport. Here, we present the first characterization of an antibody to the C-terminal of CTL1. When full length torpedo CTL1 was expressed in oocytes, a broad 60 kDa band appeared concomitant with the detection of immunoreactivity at the plasma membrane. In, the native protein was prominent throughout the CNS and along the electric nerves. CTL1 immunolabeling was particularly conspicuous in the myelin sheath surrounding the electric nerve and in central myelinated structures. The association of the presumptive choline transporter, CTL1, with myelin membranes suggests a role for this new protein in lipid production.

The immunosuppressor cyclosporin A inhibits the peptidyl-prolyl-cis/trans-isomerase activity of cyclophilins and the resulting complex inhibits the phosphatase activity of calcineurin. Both enzymes were detected in peripheral nerve endings isolated from the electric organ of Torpedo and shown to be affected by 10 micro m cyclosporin A. Among the cholinergic properties studied, choline uptake was specifically inhibited by cyclosporin A to a maximum of 40%. Cyclosporin A decreased the rate of choline transport but not the binding of the non-transportable choline analogue hemicholinium-3, indicating that the number of membrane transporters was not affected. Through the use of two other immunosuppressors, FK506, which also inhibits calcineurin, and rapamycin, which does not, two different mechanisms of choline uptake inhibition were uncovered. FK506 inhibited the rate of choline transport, whereas rapamycin diminished the affinity for choline. The Torpedo homologue of the high affinity choline transporter CHT1 was cloned and its activity was reconstituted in Xenopus oocytes. Choline uptake by oocytes expressing tCHT1 was inhibited by all three immunosuppressors and also by microinjection of the specific calcineurin autoinhibitory domain A457-481, indicating that the phosphatase calcineurin regulates CHT1 activity and could be the common target of cyclosporin and FK506. Rapamycin, which changed the affinity of the transporter, may have acted through an immunophilin on the isomerization of critical prolines that are found in the tCHT1 sequence.

It has recently been suggested that the brain-specific Na+-dependent phosphate inorganic co-transporter (BNPI) is able to support glutamate transport and storage in synaptic vesicles. A procedure for measuring the vesicular pool of glutamate is described and was used to select cell lines according to their glutamate storage capacity. Two cell lines were selected: C6BU-1, with a large intracellular glutamate storage capacity, and NG108-15, devoid of it. Their contents in BNPI mRNA were compared by RT-PCR. We found that both cell lines had BNPI, but in addition C6BU-1 alone expresses the other isoform, DNPI. We also carried out a clonal selection of NG108-15 cells in the presence of the dye Evans blue, a competitive inhibitor of vesicular glutamate transport, very toxic for cells in culture. It was assumed that only those that sequester and eliminate the drug by overexpressing a vesicular glutamate transporter would survive. We found that the NG108-15 clones resistant to Evans blue had an increased storage capacity for glutamate. These cells also up-regulated the BNPI isoform of the phosphate transporter as shown by RT-PCR and northern blot.

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3. 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.

1. Presynaptic injection of cyclic ADP-ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2. The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage-clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. 3. Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. 4. cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. 5. Ca2+ imaging with the fluorescent dye rhod-2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. 6. Ryanodine and 8-amino-cADPR, a specific antagonist of cADPR, decreased ACh release. 7. ADP-ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase-polymerase chain reaction experiments. 8. Application of NAD+, the substrate of ADP-ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8-amino-cADPR. 9. These results support the view that Ca(2+)-induced Ca2+ release might be involved in the build-up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.

Neuronal properties such as neurotransmitter uptake and release can be expressed in non-neuronal cells. We show here that fibroblasts-mouse cell line L-M(TK-)-are able to take up acetylcholine from the external medium and to release it in response to a calcium influx. Release was assessed biochemically by a luminescence method, but it was also elicited from individual fibroblasts and recorded in real-time using a Xenopus myocyte as an acetylcholine detector. After treatment for three to six days with dibutyryl-cyclic AMP, the cells changed their shape and acetylcholine release was greatly enhanced. Surprisingly, in differentiated fibroblasts the time-course transmitter release exhibited a high degree of variability even for the successive responses evoked from the same cell; many currents recorded in myocytes on electrical stimulation of fibroblasts had an extremely long duration (up to 1 s or more). This suggested that the release sites were kept open for a very long time. Cyclic AMP treatment also caused a marked increase in the expression of mediatophore 16,000 mol. wt proteolipid in fibroblast membranes. Mediatophore is an acetylcholine-translocating protein which is abundant in cholinergic presynaptic plasma membranes. It is concluded that cyclic AMP differentiation of fibroblasts prolongs the duration of acetylcholine release at individual sites and enhances the expression of the 16,000 mol. wt proteolipid-forming mediatophore.

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.

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.

Xenopus laevis oocytes were injected with poly(A)+ RNAs extracted from the electric lobes of Torpedo marmorata, which contain a homogeneous population of cholinergic neurons. These primed oocytes were able to synthesize acetylcholine and to release the neurotransmitter in a calcium-dependent manner. Fractionation of oocyte membranes as well as immunofluorescence experiments showed that the 15-kDa proteolipid, a common subunit of the vacuolar H(+)-ATPase and of a presynaptic membrane protein capable of calcium-dependent acetylcholine translocation called the mediatophore, was located at the oocyte plasma membrane. In contrast, oocytes injected with separate transcripts encoding the 15-kDa proteolipid and choline acetyltransferase were unable to release acetylcholine in spite of an equivalent acetylcholine content and a higher level of 15-kDa proteolipid expression. We observed by immunofluorescence that under these conditions, the 15-kDa proteolipid was expressed in granular cytoplasmic membranes, which were then identified as being Golgi vesicles by cell fractionation. The striking difference in the distribution of the 15-kDa proteolipid expressed in oocytes primed with Torpedo electric lobe mRNA as compared with that seen in oocytes injected with the cRNA alone suggests that another protein endogenous to the electric lobe may be implicated in the localization of the 15-kDa proteolipid at the plasma membrane. Moreover, such a targeting mechanism could contribute to the capacity of electric lobe mRNA-injected oocytes to release acetylcholine.

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.

The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 +/- 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc-17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N- and C-termini. The approximately 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV-1 fibroblast cells, possesses a high-affinity binding site for vesamicol (Kd = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.

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.

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.

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 lambda Zap 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.

The release of ATP after potassium depolarization was measured on fragments of electric tissue incubated in a solution containing the firefly extract. Light emission was proportional to the extracellular KCL concentration. In contrast to the release of ATP after single nerve impulses, the release after direct KCL depolarization was insensitive to curare of eserin.

Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence. First, the synaptic vesicle fraction is the gradient peak for calcium uptake. Second, it was not possible to separate ACh and ATP from the uptake peak after refractionation of synaptic vesicles. Third, the fraction appears very pure on morphological and biochemical grounds. The physiological significance of the calcium uptake by synaptic vesicles is discussed.

Cholinergic synaptic vesicles were purified from Torpedo electric organ to near morphological homogeneity. They were isolated in a K+ environment. A method is described for the preparation of concentrated synaptic vesicles that allows uptake studies by conventional techniques. An ATP-Mg-dependent calcium uptake associated with synaptic vesicles is characterized. The uptake system transports calcium against a high concentration gradient. The maximum accumulation rate is obtained for the calcium, Mg++ and ATP concentrations likely to be found in the nerve terminal cytoplasm. It is suggested that synaptic vesicles are implicated in the removal of the calcium entering the nerve terminal during synaptic activity.

The membrane potential of purely cholinergic synaptosomes isolated from Torpedo electric organ was monitored with fluorescent carbocyanine dyes. An increased fluorescence was associated with depolarization and a quenching with hyperpolarization. Fluorescence data provided evidence that Torpedo synaptosomes have a membrane potential mainly driven by a K+ diffusion potential and a membrane potential of about -50 mV could be estimated after calibration of fluorescence signals with ionophore antibiotics. The release of acetylcholine (ACh) from Torpedo synaptosomes was monitored continuously by measuring the light emitted by a chemiluminescent method (Israël & Lesbats, 1981 a). Using fluorescence data, the release of ACh was expressed as a function of membrane potential. The relationship between presynaptic potential and transmitter release as determined by biochemical methods at cholinergic nerve endings showed striking similarities to that observed at the squid giant synapse. Several substances were also tested with regard to their depolarizing and releasing properties and it was found that the toxin isolated from the venom of the annelid Glycera convoluta, which induced a large increase in quantal release of transmitter (Manaranche, Thieffry, & Israël, 1980) promoted a depolarization of Torpedo synaptosomes in addition to ACh release.

The release of acetylcholine (ACh) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay (Israël & Lesbats, 1981 a, b). Upon prolonged K+ depolarization in the presence of Ca2+, the release of ACh was transient and returned to a steady low level in about 3 min. Addition of the Ca2+ ionophore A23187 triggered the release again, suggesting that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response, but rather an inactivation of the Ca2+ entry. The release response evoked by adding Ca2+ back after exposure of the synaptosomes to high K+ (70 mM) and low Ca2+ (0.57 mM) solution inactivates as a function of the duration of the pre-depolarization with a two-component time course with rapid (tau = 5.5 s) and slow phases (tau = 143 s). This response to Ca2+ addition was more strikingly reduced as the level of depolarization during pre-treatment was increased. The inactivation was found to be dose dependent with respect to the amount of Ca2+ present during the pre-depolarization period (conditioning Ca2+). Moreover, the presence of EGTA during pre-treatment with high-K+ solutions increased the response to applied Ca2+. These observations suggest that Ca2+ entry itself was responsible for this inactivation. No inactivation was found when ACh release was induced by the depolarizing agent Gramicidin D, except when external Na+ was replaced by Li+. This result indicates that part of the Ca2+ influx promoted by Gramicidin D depends on a Na+ entry, and may be mediated by the Na-Ca exchange mechanism.

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.

1. When a slice of electric organ of Torpedo is stimulated and superfused with a solution containing a firefly lantern extract, it is possible to measure the release of ATP after each nerve impulse as a light emission. 2. The postsynaptic action of released ACh induces the release of ATP by the postsynaptic cell. Most of the released ATP is of postsynaptic origin. 3. Ion fluxes associated with depolarization, or depolarization itself, trigger the release of ATP from postsynaptic and presynaptic membranes (synaptosomes). 4. ATP is able to block ACh release; a postsynaptic "retrograde transmission" able to control presynaptic transmitter release is possible.

The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromaffin cells in the adrenal medulla.

The release of ACh (acetylcholine) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay. A maintained depolarization by high KCl in the presence of Ca2+ triggered only a transient ACh release. It was shown that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response. The termination of release was probably caused by inactivation of voltage-dependent Ca2+ entry and rapid removal of intraterminal Ca2+ by a (Na+)0 dependent mechanism. It was found that exposure of the synaptosomes for a short period to low Ca2+-high K+ solutions greatly reduced the responses to Ca2+ reintroduction, as compared to the control release obtained when high K+ was applied in the presence of normal Ca2+. The response to Ca2+ reintroduction was measured following various times of preincubation with high K+ and low Ca2+; thus, an estimate of the time course of the inactivation of Ca2+ permeability during a depolarization could be made. A two component exponential kinetic was observed, with a rapid (tau = 3.6 s) and a slow phase (tau = 77 s). This inactivation was more pronounced when a higher KCl concentration was used to induce a greater depolarization. The presence of EGTA during the preincubation with high KCl greatly increased the response provoked by Ca2+ reintroduction, whereas increases in Ca2+ during the preincubation period caused proportional reduction in the subsequent response to Ca2+ reintroduction, indicating that the Ca2+ influx itself was involved in the inactivation process.