Torpedo Electric Organ Research Articles

Ca2+-dependency and substrate specificity of cholinergic synaptic vesicle ATPase

The Mg(2+)-ATPase associated with highly purified cholinergic synaptic vesicles isolated from the Torpedo electric organ can be activated by millimolar concentrations of Ca(2+) only to a maximum of 46%. ATP is hydrolyzed in preference to other tri-nucleotides on Mg(2+)-activation but not on Ca(2+)-activation of the enzyme. Analysis of the Ca(2+)-dependency of vesicle associated ATPase-activities under controlled free Ca(2+)-concentrations (10(?7) to 10(?3)M) shows that vesicles do not contain a Mg(2+)-dependent, Ca(2+)-stimulated (Ca(2) + Mg(2+)) ATPase. Our results suggest that cholinergic synaptic vesicles contain a single ATPase preferentially stimulated by Mg(2+)-ions and that ATP-stimulated vesicular uptake of Ca(2+) is not due to the presence of a Mg(2+)-ATPase stimulated by micromolar Ca(2+)-concentrations.

Phylogenetic polymorphism on lectin binding to junctional and non-junctional basal lamina at the vertebrate neuromuscular junction

The histochemical binding of seven fluoreceinated lectins was comparatively studied in muscular tissue from twenty three different animal species including mammalians, amphibians, avians and fishes. Special interest was taken in the exploration of the differential lectin-binding properties at the neuromuscular synapse. Binding to synaptic sites was demonstrated using lectins that recognizes N-acetylgalactosamine and among of them, Dolichus biflorus agglutinin (DBA), was the most specific. Nevertheless, DBA fails to stain endplates in the muscle from most of the avians and the fishes (including the Torpedo electric organ) indicating that a polymorphic distribution of glycoconjugates exist at the vertebrate neuromuscular junction. Other lectins such as Concanavalin A (ConA) or Wheat germ agglutinin (WGA), share a similar staining properties in all animals that we examined making an intense label over the complete muscle surface. Although the species-related polymorphism on lectin binding does not reveal a clear relationship with the evolutionary tree, they give an evidence on the chemical heterogeneity of molecules specifically concentrated at the neuromuscular junction.

Inhibition of evoked acetylcholine release: two different mechanisms in the Torpedo electric organ

The action of agents inhibiting evoked transmitter release was investigated by analysing the Ca 2+ secretion relationship, electrophysiologically and biochemically, and by measuring the stimulation-induced 45Ca accumulation in the tissue. Transmitter release saturated at external Ca 2+ concentrations higher than 4 mM, the releasing mechanism probably being the limiting step. Antagonists of Ca 2+ entry (Mg 2+, Cd 2+, diltiazem) decreased the sensitivity of acetylcholine release to Ca 2+, acting as competitive inhibitors, and reduced the stimulation-induced 45Ca accumulation. Quinacrine produced similar effects, indicating that it interacts primarily with Ca 2+ entry. In contrast, exotremorine and adenosine depressed transmitter release proportionally at all Ca 2+ concentrations, acting as non-competitive inhibitors, and did not modify the stimulation-induced 45Ca accumulation. Their inhibitory effects were additive and reflected a decrease in the quantal content of the responses evoked. It is concluded that both drugs inhibit cholinergic transmission in the electric organ without altering Ca 2+ entry into the nerve endings.

Angiotensin-converting enzyme associated with Torpedo california electric organ membranes.

Torpedo electric organ contains high concentrations of angiotensin converting enzyme (ACE) like activity, cleaving [Leu5]enkephalin at the Gly3-Phe4 peptide bond. Most of the activity cosediments with the cell membranes. The enzymatic preparation from membranes is inhibited by low concentrations of the ACE inhibitors, SQ 14225 and SQ 20881 (IC50 of 0.6 and 15 nM, respectively), and is weakly inhibited by the neutral endopeptidase inhibitors, phosphoramidon and thiorphan (IC50 of 30 microM and ca. 70 nM, respectively). The enzyme degrades hippuryl-His-Leu and is activated by NaCl. Hippuryl-His-Leu and [Leu5]enkephalin are degraded with Km of 93 and 41 microM, and Vmax of 21 and 10 nmol/mg protein/min, respectively. The specific activity of the ACE-like activity in homogenates of Torpedo electric organ is relatively high (6.3 nmol hippuryl-His-Leu/mg protein/min); this value is similar to that obtained for rat lung and rat striatum.

Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes.

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.

Depolarization-induced release of ATP from cholinergic synaptosomes is not blocked by botulinum toxin type A

We report here the effects of Botulinum Toxin type A on the release of ATP and Acetylcholine from Torpedo electric organ synaptosomes. Our results show that Botulinum Toxin type A inhibits specifically the K(+)-induced release of Acetylcholine from synaptosomes without affecting the release of ATP. Membrane potential and calcium uptake into cholinergic nerve terminals are not modified after Botulinum Toxin poisoning. It is suggested that either most of the ATP released during the depolarization of the cholinergic synaptosomes does not originate from cholinergic synaptic vesicles or that there are two populations of synaptic vesicles, Acetylcholine-enriched synaptic vesicles and ATP-enriched synaptic vesicles. However, the possibility that the ACh and ATP released could come from different intrasynaptosomal compartments cannot be excluded.

The Torpedo electrocyte: a model system for the study of receptor-cytoskeleton interactions.

We have used the electrocyte of Torpedo electric organ as a model system for the study of AchR stabilization in the postsynaptic membrane. Attention was focused on membrane cytoskeleton interactions in particular on a peripheral protein of 43 KD that is believed to participate in AchR immobilization. Using immunocytochemical methods, we have shown that the cortical skeleton in Torpedo electrocyte displays a local differentiation proper for each specialized domain of the plasma membrane. In the postsynaptic membrane, characterized by an accumulation and a geometrical organization of the receptors in the plane of the membrane, the 43 KD protein participates in a submembraneous coating or "postsynaptic densities" that strictly codistribute with the AchR. The 43 KD protein might also account for the anchoring of intermediate-sized filaments. The organization of the postsynaptic domain appears readily different from that of the non-innervated one where the membrane folds are maintained by a cortical meshwork of cytoskeletal proteins such as ankyrin, spectrin and oligomeric actin. In conclusion, the asymmetrical organization of the cortical skeleton in the electrocyte offers a unique opportunity for the study of the specific aspects of membrane-skeleton interactions that take place in the postsynaptic domain.

Brief occurrence of a population of presynaptic intramembrane particles coincides with transmission of a nerve impulse.

Small pieces of Torpedo electric organ were cryofixed at 1-ms time intervals in a liquid medium at -190 degrees C before, during, and after the passage of a single nerve impulse. In contrast to studies using this or other preparations, these experiments were done without 4-aminopyridine or other drugs that potentiate transmitter release. Freeze-fracture replicas were made from the most superficial layers of the tissue, where the rate of cooling was rapid enough to retain ultrastructure in the absence of chemical fixation. We found that the transmission of an impulse was accompanied by the momentary appearance of a population of large intramembrane particles in both the protoplasmic (P) and the external (E) leaflets of the presynaptic plasma membrane. The change was very brief, appearing soon after the stimulus artifact. It lasted for 2-3 ms. Large pits denoting vesicle openings at the presynaptic membrane were found in a small proportion of nerve terminals; their number did not increase during transmission of the nerve impulse. Reducing the temperature from 16 to 5 degrees C slowed the time course of both the electrophysiological response and the change in intramembrane particles. The number of large particles did not increase when stimulation was applied in a low-Ca medium, a condition where the nerve terminals were still depolarized by the action potential but did not release the neurotransmitter. From these and other observations, we conclude that this transient change of intramembrane particles is closely linked to the mechanism of acetylcholine release at the nerve-electroplaque junction.

Alpha-Bungarotoxin binds to low-affinity nicotine binding sites in rat brain.

Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]alpha-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an alpha-bungarotoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatography step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]alpha-bungarotoxin to P2 membranes was inhibited by (-)-nicotine (Ki = 9 X 10(-6) M), and this sensitivity was preserved after affinity purification. It is proposed that alpha-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]alpha-bungarotoxin displayed little stereoselectivity. In this respect, the brain alpha-bungarotoxin binding site resembles the nicotinic acetylcholine receptor from Torpedo electric organ.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by α-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Patch clamp measurements onXenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Potentiation by 4-aminopyridine of quantal acetylcholine release at the Torpedo nerve-electroplaque junction.

The effects of 4-aminopyridine (4-AP) on electrophysiological post-synaptic responses evoked by field stimulation or evoked focally using a loose patch-clamp technique, and on radiolabelled transmitter release were studied in the Torpedo electric organ. In this preparation, 4-AP had three major effects: it greatly potentiated the amount of acetylcholine (ACh) released by a nerve impulse, it prolonged the duration of the post-synaptic electroplaque current (e.c.) by several hundreds of milliseconds, and it increased the delay of responses triggered by a presynaptic action potential. Noise analysis performed at different times during the focally recorded giant response showed that it was made of a sustained release of ACh quanta. The maximum synchronous release of transmitter, expressed as the maximum number of quanta simultaneously delivered/micron2 of presynaptic membrane, was apparently not modified by 4-AP. A slightly different dose dependence was found for the effects of 4-AP on the potentiation of transmitter release and on the prolongation of the synaptic delay. The effects of tetraethylammonium (TEA) and other K+ channel blockers on these parameters were similar to those of 4-AP. Strong depolarizing pulses applied focally to a nerve ending were able to evoke a giant response even in the presence of 1 microM-tetrodotoxin (TTX). The prolongation of the discharge by 4-AP was therefore not caused by repetitive re-excitation of the nerve branches. Both the amplitude and the time course of the giant response were Ca2+ dependent. At a low Mg2+ concentration, the Ca2+ dependence of transmitter release was identical in the presence or absence of 4-AP. Paradoxically, in the presence of 4-AP, addition of 4 mM-Mg2+ considerably increased the Ca2+ dependence of release, whereas in the absence of 4-AP, Mg2+ blocked transmitter release by decreasing its sensitivity to Ca2+. This potentiating interaction between Mg2+ and 4-AP was not seen with TEA or guanidine. In conclusion, 4-AP potentiates ACh release in two different ways in the Torpedo electric organ: it promotes a sustained quantal release of transmitter during several hundreds of milliseconds without any significant change in the maximal synchronous release, it interacts with Mg2+ in such a manner that the sensitivity to Ca2+ of the nerve terminals is increased.

Effects of pyrethroids on nicotinic acetylcholine receptor binding and function

The mechanism of interaction of two pyrethroids with the nicotinic acetylcholine (ACh) receptor was investigated by means of their effects on binding of radioligands to Torpedo electric organ receptor and its tracer ion flux, as well as by means of their effects on ACh sensitivity of denervated rat soleus muscle. Allethrin and fluvalinate had no effect on the equilibrium binding of 125I-labeled α-bungarotoxin (α-BGT) to the ACh receptor, but both decreased its initial rate of binding in presence of carbamylcholine (carb). In absence of carb, only allethrin decreased the rate of 125I-α-BGT binding slightly. Binding of [ 3H]perhydrohistrionicotoxin (H 12-HTX) to the channel sites of the ACh receptor was inhibited more by allethrin than by fluvalinate, but unlike H 12-HTX, neither pyrethroid had much effect on receptor-activated 22Na + influx into Torpedo microsacs, nor did fluvalinate reduce ACh sensitivity in denervated rat soleus muscle. Thus, it is suggested that the pyrethroids bind to a site that is different from the agonist or H 12-HTX binding sites. Both fluvalinate and allethrin were potent in inhibiting receptor-activated 45Ca 2+ uptake, and their effect increased with time. The RS fluvalinate isomer was more potent than the RR in inhibiting 45CA 2+ uptake, in agreement with their relative toxicities to insects. Fluvalinate and allethrin (1 μ m) increased the affinity of ACh receptors for carb at low concentrations and decreased it at high concentrations, suggesting that binding of pyrethroids affects agonist-induced conformational changes. The ACh sensitivity of denervated soleus muscle fibers was greatly depressed when the buffer was Na + free and contained 95 m m Ca 2+, but fluvalinate significantly increased the ACh sensitivity. Thus, it is suggested that the pyrethroids bind to the nicotinic ACh receptor and interfere with receptor desensitization.

Amphiphilic and hydrophilic forms of choline-O-acetyltransferase in cholinergic nerve endings of the Torpedo

In the purely cholinergic nerve endings isolated (i.e. synaptosomes) from the electric organ of the fish Torpedo, the enzyme choline acetyltransferase was found to exist not solely in its well-known soluble form but also in a form which is non-ionically bound to the plasma membrane; this activity could not be solubilized in solutions of high ionic strength (0.5 M NaCl). The non-ionic detergent Triton X-114 was used to solubilize synaptosomes isolated from either the electric organ of Torpedo or rat brain. This detergent allows to separate hydrophilic from amphiphilic proteins of cells or subcellular fractions. Twelve per cent of the synaptosomal choline acetyltransferase partitioned as amphiphilic and 80–97% as hydrophilic activity. The percentage of amphiphilic activity present in synaptosomes was significantly higher than that of the form of activity (4.4%) extracted from samples containing only the soluble form of choline acetyltransferase but was significantly lower than the percentage of amphiphilic enzyme present in preparations of synaptosomal plasma membrane (20–22%) which were enriched in the non-ionically membrane-bound form of choline acetyltransferase. These results indicate that the soluble and the non-ionically membrane-bound enzymes differ in their capacity to interact with non-ionic detergents. The preparations of synaptosomal plasma membranes contained significantly higher proportions of detergent-insoluble choline acetyltransferase activity than did the whole synaptosomes; the difference was more striking for the Torpedo than for the rat enzyme. This detergent-insoluble activity was not due to aggregates of the enzyme. Some properties of the hydrophilic and amphiphilic choline acetyltransferase of Torpedo were analyzed. The two forms of the enzyme did not exhibit different affinities for their substrates; they were found to differ with respect to their sensitivity to (i) inhibition by increasing concentrations of the two products of the reaction, acetylcholine and coenzyme A (ii) heat inactivation at 45°C. Most probably the hydrophilic and amphiphilic activities correspond to what was referred to as soluble and non-ionically membrane-bound choline acetyltransferase, respectively. The amphilic form may be an integral enzyme of the plasma membrane of cholinergic nerve endings or may be tightly bound to a specific protein in this membrane which may act as a “receptor” for choline acetyltransferase.

Ectonucleotidase activities associated with cholinergic synaptosomes isolated from Torpedo electric organ.

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.

Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction

Small pieces of Torpedo electric organ were treated with 4-aminopyridine. a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120–150 ms after the stimulus, that is a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also earned out in a modified solution containing no added calcium. 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro. transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane. At variance from the results obtained on the frog neuromuscular junction [Heuser et al. (1979) J. Cell Biol. 81, 275] the present changes of intramembrane particles apparently did not follow vesicle fusion. The possible explanations for the discrepancy between the two sets of results have been discussed.

Immunochemical and pharmacological distinctions between curaremimetic neurotoxin binding sites of central, autonomic, and peripheral origin.

Comparative pharmacological and immunochemical studies were conducted on alpha-bungarotoxin binding sites from rat brain or muscle, Torpedo electric tissue, or the TE671 or PC12 clonal cell lines. Characteristic distinctions were observed in the pharmacological profile of drugs competing for toxin binding to different tissues. Differences also were found in the proportion of toxin binding sites (membrane-bound or detergent-solubilized) that are immunologically reactive with either monoclonal antibodies directed against nicotinic acetylcholine receptors from the electric organ of Torpedo or polyclonal antisera raised against nicotinic receptors from the electric organ of Electrophorus. These results suggest that toxin binding sites are structurally heterogeneous. Structural heterogeneity of nicotinic acetylcholine receptors, neurotoxin binding sites, or both, may contribute to the manifestation of nicotinic receptor functional heterogeneity and may explain the apparent discrepancy at some sites between toxin binding activity and toxin functional potency.

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

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.

Putative Cholinergic‐Specific Gangliosides in Guinea Pig Forebrain

The nature of the cholinergic-specific antigen Chol-1 recognized by an antiserum raised against Torpedo cholinergic electromotor synaptosomal plasma membranes was investigated in guinea pig forebrain to establish whether it has a gangliosidic nature in guinea pig as in Torpedo. Gangliosides extracted from guinea pig forebrain and extensively purified to eliminate peptide contaminants were effective in inhibiting the selective lysis of the cholinergic subpopulation of cortical synaptosomes induced by the antiserum. Neuraminidase, protease, alkali, and heat treatment did not impair the inhibitory activity of gangliosides. Whereas the antiserum recognized many gangliosides from Torpedo electric organ, the immunostaining of guinea pig forebrain gangliosides separated on TLC showed only two immunopositive bands migrating close to GT1b and GQ. After affinity purification on Torpedo electric organ gangliosides the activity of the antiserum in inducing complement-mediated lysis was increased and it still recognized the two ganglioside bands on TLC. These results strongly suggest the existence of two polysialogangliosides bearing antigenic determinants specific for the cholinergic neurons.

Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release.

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.