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.     

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.     

Separation of synaptic vesicles of different functional states from the cholinergic synapses of the Torpedo electric organ

Synaptic vesicles were isolated on sucrose zonal gradients from perfused tissue blocks of Torpedo electric organ. They give rise to a coincident peak in the concentrations of acetylcholine and adenosine 5′-triphosphate. On low-frequency stimulation (0.1 Hz) of the nerve attached to the tissue block a distinct population of synaptic vesicles is found that sediments further into the density gradient forming a second (denser) vesicle peak. When dextran is added to the perfusate, only these denser vesicles contain electron-dense granules. This second (denser) peak contains about 25% of all vesicular acetylcholine and about 30% of the adenosine 5′-triphosphate and most of the newly synthesized acetylcholine as shown by incorporation of radiolabelled acetate. The specific radioactivity of acetylcholine in the denser vesicles after 1800 impulses is on average 16.5 times higher than that of the vesicles sedimenting at the original density and 9.2 times higher than the average of all vesicles isolated. The specific radioactivity of total tissue acetylcholine is lower than the average for all vesicles.It is concluded that stimulation makes apparent metabolic and morphological heterogeneity of synaptic vesicles. The increase in density of the vesicles containing newly synthesized acetylcholine could be due to endocytotic uptake of sucrose contained in the perfusate after the vesicle has undergone exocytosis. The results suggest that synaptic vesicles can be reloaded with transmitter and re-used even after uptake of extracellular marker.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Activities of alkaline phosphatase and Na+-K+-adenosine 5'-triphosphatase in the brain of experimentally produced histidinaemic squirrels (Funambulus palmarum)

In the two areas of the brain examined (olfactory lobes and cerebral hemispheres) the activity of alkaline phosphatase was lower in squirrels that had received injections of l-histidine for 4 days; the degree of inhibition of the enzyme was greater when the histidinaemia was more severe. A much smaller reduction was found in the activity of Na⁺-K⁺-adenosine 5′-triphosphatase. The possibility that these effects of histidinaemia might be related to the changes in the transport of substances into the brain that occur in this syndrome was discussed.     

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.     

Comparison of synaptic plasma membrane and synaptic vesicle polypeptides by two-dimensional polyacrylamide gel electrophoresis.

Extracts of chick brain synaptic plasma membranes, synaptic vesicles, and mixtures of membranes and vesicles were examined by electrophoresis on two-dimensional polyacrylamide gels by a modification of the O'Farrell technique. Synaptic plasma membranes had twenty-one major polypeptides; synaptic vesicles had seventeen. Thirteen major polypeptides were common to both fractions. The similarities between the synaptic vesicle and synaptic plasma membrane patterns are unlikely to be due to contamination of one fraction by the other or to contamination of both fractions by microsomes, synaptoplasm or mitochondria. Our findings are consistent with mixing of membrane proteins occurring during exocytosis but it remains to be shown that these synaptic subfractions are not contaminated by a type of membrane for which markers are not yet available.     

The uptake of ascorbic acid and dehydroascorbic acid by chromaffin granules of the adrenal medulla.

Adrenal chromaffin granules incubated with [¹⁴C]ascorbate or [¹⁴C]dehydroascorbate accumulated radioactively labeled compounds. Dehydroascorbate was generated from ascorbate by including oxidized cytochrome c in the incubation mixture but thin layer chromatography revealed that dehydroascorbate was present even in the absence of cytochrome c. This observation coupled with the fact that radioactively labeled compound was accumulated much faster in the presence of cytochromec than in its absence indicates that dehydroascorbate but not ascorbate is taken up by the chromaffin granules. Uptake of radioactivity was not saturable with ascorbate or dehydroascorbate concentration, and granules which had taken up the radioactively labeled compound lost most of the material in 12 h. In addition 1 mm adenosine 5′-triphosphate--Mg²⁺ + had no effect on either ascorbate or dehydroascorbate uptake. These data indicate that uptake proceeds via an energy independent mechanism. The rate of uptake of dehydroascorbate is 50-fold lower than the rate of catecholamine uptake.Ascorbate but not dehydroascorbate acts as a cofactor for the enzyme dopamine β-hydroxylase in the conversion of dopamine to norepinephrine. Dehydroascorbate taken up by the granules must therefore be reduced in order to be used by dopamine i3-hydroxylase. We suggest that the chromaffin granule electron transport system may be involved in this reduction.     

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.     

Changes in morphology and physiology of olfactory receptor cilia during development

Ciliated olfactory receptor neurons in vertebrates turn over throughout life. We show that these neurons bear different types of cilia at different developmental stages; cilia on newly differentiating cells are short and motile; cilia on mature cells are longer and immotile; Mg²⁺ and adenosine 5′-triphosphate are requisite for ciliary motion; stimulation with odorants can induce synchronous motion and that this process is mediated by Ca²⁺.We propose that receptor neurons have two distinguishable developmental states. In the first, before the growing axon establishes synaptic connection to the brain, the cells bear motile cilia and are generally irritable. In the second, the cilia are long and immotile and the cells can distinguish between odorants.     

Ultrastructural demonstration of adenylate cyclase activity in the rat neostriatum.

The ultrastructural histochemical localization of adenylate cyclase activity in the rat neostriatum was studied using cobalt as a precipitating agent. Basal adenylate cyclase activity was found intracellularly in some dendrites, whereas the neuronal cell bodies and glial cells were negative. In vitro stimulation of the tissue slices with dopamine before the histochemical procedure increased the adenylate cyclase activity which was also found associated with the postsynaptic membranes of axodendritic and axo-spinous synapses mainly containing small empty vesicles. Activity was also seen intracellularly in some nerve terminals containing synaptic vesicles.The significance of the cytochemical localization of dopamine-stimulated adenylate cyclase and the possible morphology of the dopaminergic synapses, including their possible interconnections, are discussed.     

Barium-induced inhibition of K+ transport mechanisms in cortical astrocytes--its possible contribution to the large Ba2+-evoked extracellular K+ signal in brain

Homogenous mouse astrocytes in primary cultures were used to investigate the action of different Ba2+ concentrations on 42K transport, membrane potential and Na+,K+-adenosine triphosphatase activity. Five millimolar Ba2+ reduced total K+ influx and efflux (each by 83%) and ouabain-sensitive net K+ uptake (by 80%); it decreased the K+ content, depolarized the membrane potential reversibly and completely inhibited the Na+,K+-adenosine triphosphatase activity. The concentration dependence of these effects was biphasic. Concentrations between 2 and 20 microM affected only the passive K+ fluxes (IC50: 6 microM). Concentrations between 50 microM and 5 mM inhibited the Na+,K+-adenosine triphosphatase and had no further effect on passive fluxes, but inhibited the ouabain-sensitive net uptake of K+ (IC50: 3.1-0.6 mM). It is suggested that the large evoked extracellular K+ increase in the brain observed in Ba2+-treated preparations in vivo or in brain slices to a large extent is due to the impairment of passive and active K+ clearance by glial cells.