Choline Uptake and Metabolism in Affinity-Purified Cholinergic Nerve Terminals from Rat Brain

Cholinergic nerve terminals were affinity purified from rat caudate nucleus. These terminals possessed both high- (KT = 2.7 microM) and low- (KT = 58 microM) affinity uptake mechanisms for exogenous [3H]choline. The proportion of [3H]choline acetylated was reduced from 75 to 30% under conditions of anoxia and hypoglycaemia, whereas the phosphorylation of choline increased from 4 to 52%. Choline phosphorylation was also increased when the terminals were preloaded with choline. The affinity-purified terminals were shown to release acetylcholine in a Ca2+-dependent manner on depolarization. The relationship between choline acetylation and phosphorylation in the cholinergic nerve terminal is discussed.     

Changes in cortical acetyl-CoA metabolism after selective basal forebrain cholinergic degeneration by 192IgG-saporin

The aim of the present study was to reveal whether reduced cortical cholinergic input affects the acetyl-CoA metabolism in cholinoceptive cortical target regions which may play a causative role for the deficits in cerebral glucose metabolism observed in Alzheimer's disease. The effect of cortical cholinergic denervation produced by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin, on activities of pyruvate dehydrogenase and adenosine triphosphate (ATP)-citrate lyase as well as on the level of synaptoplasmic and mitochondrial acetyl-CoA and acetylcholine release in cortical target regions was studied. Cholinergic lesion produced 83%, 72% and 32% decreases in the activities of choline acetyltransferase, acetylcholinesterase and ATP-citrate lyase in nerve terminals isolated from rat brain cortex, respectively, but no change in pyruvate dehydrogenase activity. Spontaneous and Ca2+-evoked acetylcholine release from synaptosomes was inhibited by 76% and 73%, respectively, following immunolesion. The lesion-induced 39% decrease of acetyl-CoA level in synaptosomal mitochondria was accompanied by 74% increase in synaptoplasmic fraction. Levels of acetyl-CoA and CoASH assayed in fraction of whole brain mitochondria from lesioned cortex were 61% and 48%, respectively, higher as compared to controls. The data suggest a preferential localization of ATP-citrate lyase in cholinergic nerve terminals, where it may contribute to the transport of acetyl-CoA from the mitochondrial to the cytoplasmic compartment. They provide evidence on differential distribution of acetyl-CoA in subcellular compartments of cholinergic and non-cholinergic nerve terminals. There are also indications that cholinergic activity affects acetyl-CoA level and its intracellular distribution in glial and other non-cholinergic cortical cells.     

A Dopaminergic Cell Line Variant Resistant to the Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Cholinergic nerve terminals were affinity purified from rat caudate nucleus. On stimulation with both 22.6 mM KCl and 50 microM veratridine, ATP was released in a Ca2+-dependent manner. The molar ratio of released acetylcholine to ATP (9:1) was closer to that found in isolated cholinergic vesicles (7:1) than whole terminals (3:1). Extracellular [14C]ATP was rapidly metabolized by these terminals to adenosine and inosine via ectonucleotidases. The terminals had a saturable, high-affinity uptake mechanism for adenosine (Km = 16.6 microM). Veratridine stimulation also caused the Ca2+-dependent release of nucleosides in a dipyridamole-sensitive manner. Both theophylline treatment and inhibition of extracellular ATP breakdown resulted in increased ATP and nucleoside release. Extracellular adenosine was shown to inhibit acetylcholine release, probably via the A1 receptor. The role of extracellular purines at the cholinergic nerve terminal is discussed.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

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 immunocytochemical 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.     

Cholinergic Surface Antigen Chol-1 Is Present in a Subclass of VIP-Containing Rat Cortical Synaptosomes

The colocalization of vasoactive intestinal polypeptide (VIP) with the cholinergic specific surface antigen Chol-1 was investigated in synaptosomes derived from the rat cerebral cortex. Immunoaffinity purification of cortical synaptosomes using antisera to Chol-1 resulted in the copurification of VIP and cholinergic nerve terminals. VIP was purified with a yield of 75% of that of choline acetyltransferase (ChAT). These results suggest that approximately 53% of the cortical cholinergic terminals contain VIP, whereas 75% of the cortical VIP content is present in these cholinergic terminals. Both hypotonic lysis and depolarization of the nerve terminals resulted in the differential release of VIP and acetylcholine (ACh), indicating the different compartmentalization in the same nerve terminal. Complement-mediated lysis of cholinergic nerve terminals, using antisera to Chol-1, resulted in the release of 64% of the ChAT, 71% of ACh, and 27% of the VIP. The application of our method enables quantifying and mapping, with a fast, efficient, and specific technique, the coexisting peptides in cholinergic neurons of distinct brain areas.     

Specific immunolysis of serotonergic nerve terminals using an antiserum against tryptophan hydroxylase

An antiserum to tryptophan hydroxylase purified from whole rat brain when incubated with rat striatal synaptosomes in the presence of complement caused release of 18% of LDH, 20% loss of potassium and 60% loss of tryptophan hydroxylase. Uptake of 5-HT was reduced by 60%. Anti-tryptophan hydroxylase alone, or complement alone were without action. The antiserum plus complement had no effect on DA uptake and did not release TH or GAD. These results suggest selective lysis of serotonergic nerve terminals had occurred. The antiserum plus complement reduced choline uptake by 45%. However, this did not seem due to lysis of cholinergic terminals, as ChAT was not released.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.     

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000-150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron-specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle-free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergic-specific. The function of the Mr 86,000 antigen remains to be identified.     

Partial purification of α-glycerotoxin,a presynaptic neurotoxin from the venom glands of the polychaete annelid glycera convoluta

The venom secreted from glands appended to the jaws of Glycera convoluta, a Polychaete Annelid, increases the spontaneous quantal release of transmitter from nerve terminals. The component that is biologically active on vertebrate cholinergic nerve terminals has recently been shown to be a high molecular weight protein. In the present work, the crude extract from the venom apparatus was shown to be toxic for mammals and crustaceans. It was fractionated by gel filtrations and ion exchange chromatographies. The biologically active component at frog neuromuscular junctions, α-glycerotoxin, was purified more than 1,000-fold. It is distinct from the components that are toxic for crustaceans. Purified α-glycerotoxin is a globular protein of 300,000 ± 20,000 mol wt. It has a Stokes radius of 65 Å and a sedimentation coefficient of 11 S. By its molecular properties, α-glycerotoxin appears distinct from other neurotoxins such as α-latrotoxin, which also trigger transmitter release.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Immunochemical purification and characterization of ovine alpha-fetoprotein.

Ovine alpha-fetoprotein was successfully isolated from fetal sheep serum by using rabbit anti-ovine alpha-fetoprotein linked to an agarose immunoadsorbent column. Antibody used in this affinity chromatography column was produced by immunizing a rabbit with highly purified alpha-fetoprotein-antibody complex to yield a monospecific antiserum to ovine alpha-fetoprotein. Following affinity chromatography, alpha-fetoprotein was further purified by preparative polyacrylamide disc gel electrophoresis ultimately yielding a 105-fold purification. The purified alpha-fetoprotein was homogeneous on analytical polyacrylamide disc gel electrophoresis. Ovine alpha-fetoprotein was found to be immunochemically related to human alpha-fetoprotein and to exhibit a molecular weight and amino acid composition similar to other mammalian alpha-fetoproteins.     

CHOLINERGIC NERVE ENDINGS IN OCTOPUS BRAIN

—Optic lobes of the brains of Octopus dofleini werehomogenized hasaline–sucrose medium and subjected to differential and density gradient centrifugation. The fractions and subfractions thus obtained were analysed for acetylcholine (ACh) (bioassay) and protein content and were subjected to electronmicroscopy. Bound ACh was associated with particulate fractions, and a large portion of it could be recovered in subfractions that contained predominantly nerve endings. Expressed in terms of amount of ACh per mg protein, the ACh content of the nerve ending fractions was nearly 100 times greater than that of corresponding fractions previously obtained by others from mammalian brain. Calculations show that this was the minimal amount of ACh to be expected if the isolated nerve endings were predominantly cholinergic. Octopus brain tissue is in therefore very promising for future studies on ACh metabolism and compartmentation cholinergic nerve endings.     

ASSOCIATION OF SYNTAXIN WITH SNAP 25 AND VAMP (SYNAPTOBREVIN) IN TORPEDO SYNAPTOSOMES

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2–5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H⁺ ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses. Copyright © 1996 Elsevier Science Ltd     

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.     

Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals

Antibodies were raised in rabbits to synaptic vesicles purified to homogeneity from the electric organ of Narcine brasiliensis, a marine electric ray. These antibodies were shown by indirect immunofluorescence techniques to bind a wide variety of nerve terminals in the mammalian nervous system, both peripheral and central. The shared antigenic determinants are found in cholinergic terminals, including the neuromuscular junction, sympathetic ganglionic and parasympathetic postganglionic terminals, and in those synaptic areas of the hippocampus and cerebellum that stain with acetylcholinesterase. They are also found in some noncholinergic regions, including adrenergic sympathetic postganglionic terminals, the peptidergic terminals in the posterior pituitary, and adrenal chromaffin cells. They are, however, not found in many noncholinergic synapse-rich regions. Such regions include the molecular layer of the cerebellum and those laminae of the dentate gyrus that receive hippocampal associational and commissural input. We conclude that one or more of the relatively small number of antigenic determinants in pure electric fish synaptic vesicles have been conserved during evolution, and are found in some but not all nerve terminals of the mammalian nervous system. The pattern of antibody binding in the central nervous system suggests unexpected biochemical similarities between nerve terminals heretofore regarded as unrelated.     

Cholinergic-specific glycoconjugates

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylatedN-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with aTorpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.Abbreviations AP affinity purified - ATPase adenosine 5-triphosphate phosphohydrolase - cer ceramido - ChAT choline acetyltransferase - Chol-1, –2 cholinergic-specific antigens - DA dopamine - DOG deoxyglucose - ELISA enzyme-linked immunosorption assay - EOD electric organ discharge - FAB fast atomic bombardment - GABA -aminobutyrate - GAG glycosaminoglycan - gal galactosyl - gaINAc N-acetylgalactosaminyl - glc glucosyl - Glu glutamate - 5-HT 5-hydroxytryptamine - LDH lactate dehydrogenase - NA noradrenaline - NGF nerve growth factor - S, S-S mono-, disialyl - SPM synaptosomal plasma membrane - TH tyrosine hydroxylase - TLC thin-layer chromatography - TSM Torpedo electromotor synaptosomal membrane - VIP vasoactive intestinal polypeptide - VPG vesicle proteoglycan Special issue dedicated to Dr. Leon Wolfe.     

Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

Isolation of Hippocampal Synaptosomes on Percoll Gradients: Cholinergic Markers and Ligand Binding Sites

The S1 Percoll procedure, devised empirically for cortical tissue, provides highly purified, functionally viable synaptosomes on a four-step Percoll gradient. Here, for the first time, the procedure has been applied to rat hippocampus, and the gradient fractions have been analysed with respect to cholinergic markers and the synaptosomal index, lactate dehydrogenase. The presynaptic cholinergic markers choline acetyltransferase and [3H]choline uptake were most enriched in fraction 4. In contrast, acetylcholinesterase activity was broadly distributed across the gradient, consistent with the separation of synaptic plasma membranes (in fractions 1 and 2) from synaptosomes (in fractions 3 and 4). This is supported by the recovery of muscarinic binding sites labelled with [3H]quinuclidinylbenzilate in fractions 1 and 2. (-)-[3H]-Nicotine binding sites, however, were most enriched in fraction 4, consistent with their predominantly presynaptic localisation in the CNS. These results demonstrate the applicability of the S1 Percoll method to discrete brain regions for the recovery of homogeneous and viable synaptosome fractions. The separation of presynaptic terminals from post-synaptic membranes is a further advantage of this technique.     

PARTIAL PURIFICATION, PROPERTIES AND GLYCOPROTEIN NATURE OF ARYLSULPHATASE B FROM SHEEP BRAIN

Abstract— A simple method has been developed for the partial purification of arylsulphatase B from sheep brain. This includes concanavalin A-Sepharose affinity chromatography and ionic strength-dependent binding and dissociation of the enzyme with Dextran Blue; by these methods the enzyme was purified 1344-fold with 10% recovery. The partially purified enzyme was shown to be a glycoprotein and its kinetic properties were compared with that of purified arylsulphatase A from the same source.