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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: 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 μM; in the presence of Ca2+, 66 μM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of P/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.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 30 (1978), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [3H]acetate as a precursor of ACh. Little vesicular [3H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [3H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [3H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.
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  • 3
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: During early postnatal development of the kitten visual cortex the ectoenzyme 5′-nucleotidase undergoes a characteristic redistribution. Until about postnatal week 6 it is essentially confined to synaptic contacts in input layer IV and its expression is related to the use-dependent segregation of thalamic afferents into ocular dominance columns. Subsequently, 5′-nucleotidase becomes distributed uniformly throughout all layers and is then associated selectively with glial cells. Here we describe an age-dependent alteration in the expression of a carbohydrate epitope of 5′-nucleotidase which correlates with the developmental change of the enzyme's localization. We have isolated 5′-nucleotidase from the occipital cortex of kittens of varying age and from adult cats and investigated by immunoblotting the association of the HNK-1 carbohydrate epitope with the protein. 5′-Nucleotidase carries the HNK-1 epitope in kittens of 3–9 weeks but the epitope is absent from 12-week-old kittens or adult cats. Thus, the appearance of the HNK-1 epitope correlates with the transient localization of the enzyme at synapses. The HNK-1 carrying 5′-nucleotidase may be involved in synaptogenesis and use-dependent modifications of synaptic connections.
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low-frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate-dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extra cellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low-frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using α-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin I.
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract— The effect of stimulating the electric organ of Torpedo marmorata, anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.
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  • 7
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co-release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P-40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co-release with acetylcholine or ATP.
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9–12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immuno-cytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.
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  • 9
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In pigs there are two types of Peyer's patches in the small intestine; discrete patches in the jejunum (jejPP) and a continuous patch in the terminal ileum (ilPP). The ilPP was resectioned or transposed into the upper jejunum. After the operation the size of the remaining jejPP showed no compensatory growth in either group within 10 months. However, the number of CD8+ lymphocytes in the blood, spleen, mesenteric lymph nodes, tonsils, and Peyer's patches and the number of CD4+ cells in the spleen and tonsils was reduced in comparison to those of agematched control pigs. Autologous blood lymphocytes were labelled with fluorescein isothiocyanate and retransfused. In control animals the mid-portion of the ilPP showed a lower entry of lymphocytes and the migration pattern of lymphocyte subsets was different in the animals with resectioned or transposed ilPP as compared to controls. Thus, the removal of the ilPP (about 60% of all small intestinal PP) did not result in the remaining patches adapting their size, but it did influence other lymphoid organs.
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  • 10
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: —During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.
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