Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: Effect of stimulation-induced transmitter release

Cholinergic synaptic vesicles were isolated from the Torpedo electric organ by a combination of differential and density gradient centrifugation. Iso-osmolar solutions of glycine or NaCl were used as homogenization and preparation media. The metal content of intact tissue and subcellular fractions were determined by atomic absorption spectroscopy. In the synaptic vesicle fraction ratios of metals to acetylcholine (g atom/mol) were: Na, 0.30; K, 0.10; Mg, 0.07; Ca, 0.28. Filtration of isolated vesicles revealed that the strength of metal binding depends on the ionic potential of the metal cation. Thus alkali metal ions are bound to synaptic vesicles less tightly than alkaline earth metals. Incubation of vesicles with elevated levels of NaCl led to a partial exchange of Na with K but external concentrations of CaCl₂ in the physiological range were without effect on vesicle metal ion content. Stimulation of the electric organ in vivo (5000 impulses, 5 Hz) caused a depletion of the acetylcholine and adenosine 5′-triphsophate content of the vesicles whereas the levels of metal ions were increased.It is suggested that the release of acetylcholine from synaptic vesicles exposes free negative charges to which extracellular metal cations can bind in ion exchange.     

Changes in the biochemical and biophysical parameters of cholinergic synaptic vesicles on transmitter release and during a subsequent period of rest

Synaptic vesicles were isolated on sucrose density gradients from perfused blocks of Torpedo electric organ under varying experimental conditions. Newly synthesized acetylcholine was labelled with [³H]acetate in order to distinguish synaptic vesicles which had been recycled during stimulation-induced transmitter release and, in consequence, had become smaller and denser from the larger, lighter vesicles characteristic of unstimulated tissue. After 1800 pulses at 0.1 Hz, the density of these smaller vesicles increased from a pre-stimulation value of 1.056 g.ml^?1 to 1.067 g.ml^?1, whereas their water space (measured as the space occupied by the permeant solute glycerol), decreased by 34% from 65% to 43% of vesicle volume. During a subsequent 12 h rest period, these changes were partially reversed; water space returned to 52% of control and this change was highly correlated with a decrease in vesicle density and an increase in vesicular acetylcholine. The diameter of vesicles in whole tissue sections showed corresponding changes. An additional 12 h rest period did not lead to further significant recovery, suggesting that the preparation had a limited suitability for following long term processes depending on normal energy metabolism.The results can be explained on the assumption that when vesicles reform after releasing transmitter their core has a lower osmotic pressure than that of fully loaded vesicles. Reloading is accompanied by osmotically induced rehydration.     

Purification of small dense vesicles from stimulated Torpedo electric tissue by glass bead column chromatography

Synaptic vesicles were isolated from perfused tissue blocks of Torpedo electric organ using sucrose density gradient centrifugation in swing-out rotors. After application of [³H]acetate and low frequency stimulation (0.1 Hz) a denser peak of [³H]acetylcholine could be separated from the main and coincident peak of the vesicle constitutents acetylcholine and adenosine 5'-triphosphate in accordance with previous findings using zonal centrifugation (Zimmermann & Denston, Neuroscience2, 715–730, 1977). Further separation of subcellular particles sedimenting in the range of synaptic vesicles, by chromatography through columns of porous glass beads, yielded three main fractions which were eluted in the order, large (esterase-containing) membrane particles in the void volume, larger synaptic vesicles containing acetylcholine of low specific radioactivity (peak I) and smaller vesicles containing acetylcholine of higher specific radioactivity (peak II). After stimulating the electric tissue (which causes the appearance of a large proportion of synaptic vesicles about 25% smaller in diameter; Zimmermann & Denston, Neuroscience2, 695–714, 1977), the peak of larger vesicles (peak I) also contains vesicles of smaller diameter. The glass bead column thus separates membrane fragments from synaptic vesicles, but only partially resolves larger and smaller vesicles. This is also reflected by the decrease in the ratio of the specific radioactivity of acetylcholine of peak I to that of peak II, from 8.2 for unstimulated control to 4.0 for stimulated tissue.The results demonstrate that using glass bead chromatography the smaller vesicles, which appear on stimulation-induced transmitter release, contain acetylcholine of high specific radioactivity and can be completely separated from any membrane contaminants which could possibly contain a pool of nonvesicle-bound acetylcholine.     

The isolation of pure cholinergic nerve terminal sacs (T-sacs) from the electric organ of juvenile Torpedo.

The problem of synaptosome formation in the electric organ of Torpedo has been re-investigated using tissue from juvenile fish. This tissue is softer than adult material and can be easily homogenized in an Aldridge-type homogenizer. Homogenates so prepared contain a significant number of synaptosome-like structures which can be purified by differential and density gradient centrifugation. The purified particles are enriched in acetylcholine and choline acetyltransferase; they also contain lactate dehydrogenase activity, most of which is in an occluded form. The structure of these particles as revealed by electron microscopy is unusual in that they have no post-synaptic adhesions, relatively few synaptic vesicles and no intraterminal mitochondria. Because of their unusual morphology we have named these particles nerve terminal sacs (T-sacs). A high-affinity, hemicholinium-3 sensitive choline uptake system with an apparent K_m of 1–3 μm is associated with the T-sacs.     

Recycling of synaptic vesicles in the cholinergic synapses of the Torpedo electric organ during induced transmitter release.

Blocks of innervated Torpedo electric tissue can be perfused in vitro and remain functioning for more than 24 h. Low frequency stimulation (0.1 Hz) of the attached nerve leads to a decay in electrical response and tissue content of acetylcholine although the number of vesicles counted in terminals does not fall. Stimulation leads to the appearance of a population of vesicles about 25% smaller in diameter than normal. If dextran (Molecular weight 10,000–40,000) is added to the perfusate an increasing number of vesicles becomes labelled during stimulation. After 720–1800 impulses 74% of all vesicles are found to contain dextran in their lumen. The label is contained mainly in the new small vesicle population. Perfusion with dextran per se does not lead to significant fine structural changes or uptake of dextran. After stimulation, dextran-containing vesicles are also found in the unmyelinated part of the terminal axon.It is concluded that stimulation-induced transmitter release is accompanied by a recycling of synaptic vesicles which leads to uptake of extracellular marker into the lumen of the vesicles. Thus synaptic vesicles become heterogeneous as a result of their previous exo- and endocytotic activity.     

Cholinergic synaptic vesicles isolated from Torpedo marmorata: demonstration of acetylcholine and choline uptake in an in vitro system

The transport properties of highly purified synaptic vesicles, isolated from the purely cholinergic electric tissue of Torpedo marmorata, were studied in an in vitro system using column chromatography. Under iso-osmotic conditions, which did not result in substantial loss of endogenous acetylcholine or adenosine 5′-triphosphate, radiolabelled acetylcholine and choline were accumulated into vesicles (and were releasable by hypo-osmotic shock) by a time- and temperature-dependent process. Acetylcholine uptake was saturable with a K_T for transport of 3.13mM; this was competitively inhibited by choline, with a K_Tapp of 8.33 mM. Choline itself was also transported with a K_T of 9.99mm. The facilitated uptake of both compounds was specifically inhibited by hemicholinium-3, but not the nonsaturable uptake seen at 0°C. Uptake of both compounds was inhibited by NaCl at concentrations above 2 mM. In double label experiments the rate of glucose penetration (identical with that of 2-deoxy-d-glucose) was found to be approximately 15–30-fold less than that of choline or acetylcholine. The accumulation of [¹⁴C]glucose remained linear at all concentrations used, at both 0°and 26°C. After 1 h incubation at 26°C the vesicular volume contained between 3 and 6 times the concentration of labelled acetylcholine and choline compared to that in the surrounding medium. The glucose concentration in the vesicles, however, remained well below that in the medium. Vesicles mechanically or chemically treated to release their soluble contents appeared to have a much reduced capacity to take up acetylcholine, and in most cases, choline also.These results demonstrate the existence in synaptic vesicles of a mechanism for the uptake and retention of acetylcholine (possibly against extremely high concentration gradients) which is not specific enough to exclude choline, at least as a pharmacological agonist in vitro.     

Kinetics of acetylcholine recovery in Torpedo electromotor synapses depleted of synaptic vesicles

Isolated fragments of Torpedo electric organ perfused with choline-free Ringer and stimulated by application of up to 1500 pulses at 1 Hz lost 58% of their synaptic vesicles and 65% of their acetylcholine. The stimulated terminals showed ultrastructural changes of varying intensity which were reversed during rest. The numbers of synaptic vesicles returned to control values by 5 h and total acetylcholine reached the control levels only after 20 h incubation in medium containing excess of choline. Net synthesis of acetylcholine as well as acquisition of acetylcholine by the reformed vesicles followed first order kinetics with respect to the degree of unsaturation of the total acetylcholine pool and the vesicular compartment, respectively.A possible interpretation of these results is that the degree of depletion of the vesicular compartment may regulate the rate of net acetylcholine synthesis, and that the vesicular compartment may account for at least 80% of total acetylcholine in the terminals.     

Release of adenosine 5′-triphosphate from synaptosomes from different regions of rat brain

The release of adenosine 5′-triphosphate by elevated extracellular concentrations of KC1 and by veratridine was determined in synaptosomal fractions prepared from different regions of rat brain. Following correction for yields of synaptosomes from the various regions, the relative distribution of K⁺-induced release was corpus striatum > cerebral cortex > medulla > hypothalamus > cerebellum. In contrast, the relative distribution of veratridine-induced release of adenosine 5′-triphosphate was medulla > corpus striatum > hypothalamus > cerebral cortex > cerebellum.From these findings, it was concluded that (1) depolarization-induced release of adenosine 5′-triphosphate was not distributed uniformly throughout the brain but varied from region to region, (2) the K⁺-induced release of adenosine 5′-triphosphate which is Ca²⁺-dependent, had a different regional distribution than the veratridine-induced release, which is greatest in Ca²⁺-free medium, and (3) the distribution of K⁺-induced release of adenosiae 5′-triphosphate did not correlate well with the known distribution of noradrenaline concentrations in rat brain, but did correlate to some extent with the distributions of 5-hydroxytryptamine, dopamine and especially acetylcholine, so that co-release of adenosine 5′-triphosphate with these transmitters may possibly occur.     

Vesicular storage and release of a false cholinergic transmitted (acetylpyrrolcholine) in the Torpedo electric organ.

The choline analogue N-[Me-³H]N-hydroxyethyl-pyrrolidinium (pyrrolcholine) was studied in the Torpedo electric organ. Pyrrolcholine is transported into isolated nerve terminal sacs by the choline high affinity uptake system (K_T = 5.7 μm). If blocks of electric tissue are perfused in the presence of both [³H]pyrrolcholine and [¹⁴C]choline both compounds become acetylated and are taken up into synaptic vesicles. This process is enhanced by low frequency stimulation (~0.1 Hz). Upon subsequent stimulation of the nerve at 5 Hz both acetylpyrrolcholine and acetylcholine are released into the perfusate by a calcium-sensitive mechanism; their molar ratio in the perfusate is the same as that for their loss from the vesicle fraction. Acetylpyrrolcholine is a potent agonist on the frog rectus abdominis muscle although 12.7-fold less active than acetylcholine.We conclude that pyrrolcholine is a precursor of a cholinergic false transmitter in the Torpedo electric organ. Acetylpyrrolcholine has been shown to fulfil all the criteria for the definition of a false transmitter, including storage in synaptic vesicles.     

Purine salvage at the cholinergic nerve endings of the Torpedo electric organ: the central role of adenosine

The uptake and metabolism of adenosine, adenine, inosine and hypoxanthine were studied at the cholinergic nerve endings of the Torpedo electric organ. In isolated synaptosomes there is a linear uptake (measured up to 60 min) for adenosine and adenine at concentrations of 0.3 μM Uptake of adenosine exceeds that of adenine by a factor of 10. Adenosine is transported into synaptosomes via a saturable uptake system (K_m, 2 μM;V_max, ～- 30 pmols/min/mg protein). 2′-Deoxyadenosine is a competitive inhibitor of synaptosomal adenosine uptake. The nerve terminal possesses anabolic pathways for the formation of adenosine 5′-triphosphate from both adenosine and adenine. Adenosine becomes phosphorylated rapidly after entry into synaptosomes to form adenosine 5′-monophosphate; adenosine 5′-diphosphate and adenosine 5′-triphosphate were also major metabolites (70%). Adenine, inosine and hypoxanthine first accumulate in the synaptosomes. However, adenine leads to major formation of nucleotides (41% adenosine 5′-triphosphate after 60 min). Only traces of adenosine-3′:5′ cyclic monophosphate are formed from both adenosine and adenine. If adenosine 5′-triphosphate is added to a suspension of intact synaptosomes it becomes degraded to adenosine.We conclude that cholinergic nerve endings in the Torpedo electric organ possess an effective purine salvage system. Adenosine 5′-triphosphate released from either a pre- or a postsynaptic source would become degraded to adenosine in the extra-cellular medium and be re-used via an uptake system for renewed synthesis of adenosine 5′-triphosphate in nerve terminals.     

Turnover of adenine nucleotides in cholinergic synaptic vesicles of the Torpedo electric organ

Synaptic vesicles were isolated from perfused blocks of electric tissue on sucrose density gradients in a zonal rotor. In vesicles from control tissue the composition was ATP (83%), ADP (15%) and AMP (2%): the corresponding figures for stimulated tissue were 69, 22 and 6% respectively: thus ATP is the predominant vesicular adenine nucleotide in both types of vesicle. Stimulation of the nerves to the tissue at a frequency (0.1 Hz) which does not cause a fall in vesicle numbers induces an approx. 50% loss of total vesicular nucleotides, the same as the degree of loss of acetylcholine in previous experiments.When tissue blocks are perfused with a solution containing [2-³H]adenosine for several hours, 85% of the radiolabel recovered in the isolated vesicle fraction is in the form of ATP. Besides some radiolabelled ADP and a reproducible but small contribution of inosinemonophosphate, traces of radiolabelled AMP, adenosine, adenine, hypoxanthine and inosine were detected. On stimulation of nerves to tissue blocks at 0.1 Hz two populations of synaptic vesicles can be isolated, the denser one of which contains the bulk (70%) of the newly synthesized vesicular ATP as well as acetylcholine. Vesicles sedimenting at the original sucrose density lose both ATP and acetylcholine. The specific radioactivity of ATP in the denser vesicles after a simulation of 1280–1800 impulses was about four times higher than that of vesicles equilibrating at the original sucrose density.The results suggest that adenosine is an effective precursor of vesicular adenine nucleotides. On stimulation nucleotides are lost from synaptic vesicles together with the neurotransmitter. The new population of vesicles appearing on stimulation has a high turnover rate for both ATP and acetylcholine.     

The water spaces in cholinergic synaptic vesicles from torpedo measured by changes in density induced by permeating substances

The water spaces in cholinergic synaptic vesicles isolated from the electromotor terminals of Torpedo marmorata electric organ have been determined as a fraction of the total vesicle volume by measuring the density changes induced in the vesicles by the addition of permeating substances to iso-osmotic density gradients. Three permeating substances were selected for study: deuterium oxide, dimethylsulphoxide and glycerol. The water spaces measured by these three substances were not equal, being 83%, 72% and 65% of the vesicle volume, respectively. When vesicles were lysed in dilute (10 mM) Hepes buffer (pH 7.0), the deuterium oxide space was not detectably changed, but the dimethylsulphoxide and glycerol spaces assumed the same value of 74–75%. This was interpreted to mean that lysis resulted in the loss of highly hydrated core constituents (presumably mainly acetylcholine and adenosine 5'-triphosphate) whose bound water can be replaced by dimethylsulphoxide and deuterium oxide but not by glycerol. When intact or lysed vesicles were exposed to highly hyperosmotic CsCl gradients, the changes in the density and in the deuterium oxide and glycerol spaces showed that the vesicles had undergone collapse due to osmotic dehydration; 91–96% of the glycerol space is osmotically active water. The density of the membrane was estimated to be 1.11 to 1.135 depending on its protein content.These results confirm, by an independent method, conclusions already reached in this laboratory from the protein and lipid analysis of vesicles (Ohsawa, Dowe, Morris & Whittaker, Brain Res.161, 447–457, 1979) and from density measurements at varying osmotic pressures (Breer, Morris & Whit-taker, Eur. J. Biochem.87, 453–458, 1978), namely, that the vesicle is a highly hydrated structure with a diameter of 80–100 nm and a lipid-rich membrane 4–5 nm in thickness.The implications of the results for measurements of vesicular membrane potential and intravesicular pH are discussed.     

Ultrastructure of the nerves in veins from human omentum

To study the ultrastructural characteristics of the sympathetic nerve terminals of human omental veins, and of their relationship to the innervated smooth muscle cells, biopsy specimens were taken during abdominal surgery, rapidly fixed in glutaraldehyde/osmium and stained with uranylacetate. The results indicate that the veins have an extensive noradrenergic innervation, penetrating into the tunica media. The distance between nerve terminals partly or wholly free from enveloping Schwann cells, to the surface of smooth muscle cells ranged from 30 to 500 nm. Large dense core vesicles were prominent in both preterminals and terminal regions, while small dense core vesicles occurred mainly in terminals. Large dense core vesicles in close contact with the axolemma were occasionally observed, indicating involvement in secretion by exocytosis.     

Nucleotide uptake by isolated cholinergic synaptic vesicles: Evidence for a carrier of adenosine 5'-triphosphate

The in vitro uptake of [³H]nucleotides was studied using cholinergic syaptic vesicles isolated from Torpedo electric organ, with a resting membrane potential of 50–60 mV. The osmotically sensitive uptake of [³H]adenosine 5'-triphosphate (ATP) was markedly influenced by temperature and external pH, and was maximal after 40–50 min; longer incubation resulted in loss of accumulated radiolabel. Similar characteristics were also observed for adenosine 5'-mono- and diphosphate and guanosine and uridine triphosphates, all of which acted as competitive substrates for the saturable system which transported ATP (K_T 1.15 mM). Breakdown of [³H]nucleotides in the medium was not a significant factor, and adenosine, guanosine and adenine were very poorly incorporated. Under conditions of V_max, vesicle to medium ratios of [³H]ATP of 20–25 were observed; the amount of radiolabel was equivalent to 20–50% of the initial endogenous amount of ATP in the vesicles. Atractyloside specifically inhibited nucleotide transport with no modification of hemicholinium-3 sensitive acetylcholine uptake. Antisera raised (a), to whole Torpedo vesicle extract, and (b), to a single purified vesicle polypeptide, greatly stimulated ATP uptake without effect on simultaneous influx of either acetylcholine or glucose.It is concluded that isolated vesicles contain a nucleotide carrier of wide pharmacological specificity (possibly the 34,000 molecular weight protein of Stadler & tashiro [1979]), which is likely to be of physiological relevance. Implications for vesicular refilling mechanisms are discussed.     

Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological and biochemical properties of synaptic vesicles isolated from guinea pig brain.

Two major types of different synaptic vesicles were isolated by centrifugation from the brain homogenate of guinea-pigs. The size and shape of vesicles in each type are different. The one type is spheroid and the other is flattened. The former is larger than the latter. These two types are equivalent to the S-type and F-type vesicles in tissue sample. Sedimentation coefficients of S-type and F-type are 64s and 59s respectively. Amino acid composition of vesicles in each type is almost similar, but the content of amino acids in the S-type is larger than in the F-type except for valine.     

The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission

The twitch response observed during low frequency electrical stimulation of postganglionic cholinergic neurones supplying the longitudinal smooth muscle of the guinea-pig ileum was markedly reduced by incubation with an homogeneous preparation of botulinum type A neurotoxin (4.3-8.6 nM). This intoxication of the autonomic cholinergic neurones was long-lasting, irreversible by washing, but readily reversed by 4-aminopyridine (50-1000 microM). The noradrenergic motor response of the rat anococcygeus following field stimulation was partially antagonised by the neurotoxin. The non-adrenergic inhibitory response of the guinea-pig taenia coli, elicited by field stimulation, was not antagonised by botulinum toxin, suggesting that a source of a non-adrenergic inhibitory transmitter exists, other than intramural cholinergic neurones. However, the neurogenic excitatory responses of the guinea-pig bladder, elicited by field stimulation in the presence of atropine and guanethidine, were virtually abolished by botulinum toxin. It is suggested that the parasympathetic neurones which supply the smooth muscle of the guinea-pig urinary bladder co-release acetylcholine and a non-cholinergic excitatory transmitter; ATP or polypeptides are possible candidates.