Tetsufumi Ueda

Glutamate accumulation into synaptic vesicles.

Publisher Summary This chapter discusses the glutamate accumulation into synaptic vesicles. A critical, but rate-limiting step, in the study of glutamate transport into synaptic vesicles is the preparation of synaptic vesicles that are free of contamination yet retain functional capability. However, this requires rather large quantities of affinity-purified antibodies to a synaptic vesicle protein, synapsin I, and, despite this requirement, the yield of such a vesicle preparation is too low to permit the analysis of vesicular glutamate uptake in small tissues. The chapter describes the assay and properties of the glutamate transport system in the synaptic vesicle in three preparations: (1) bovine brain synaptic vesicles highly purified by the use of antisynapsin I immunoglobulin G (IgG), (2) a bovine brain synaptic vesicle fraction purified by sucrose density gradient centrifugation, and (3) a rat brain crude synaptic vesicle fraction. Beyond the characterization of the properties of glutamate uptake into synaptic vesicles, little research has investigated in vivo changes in vesicular uptake.

Diminished Specific Activity of Cytosolic Protein Kinase C In Sciatic Nerve of Streptozocin-Induced Diabetic Rats and Its Correction by Dietary myo-Inositol

The impaired Na+-K+-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na+-K+-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na+-K+-ATPase activity and blunted regenerative response in diabetic nerve.

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [3H]l‐glutamate (Glu) content within the synaptosome (a pinched‐off nerve ending preparation) leads to a change in the amount of exocytotically released [3H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane‐permeant inhibitor (Ki = 19 nm) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H+‐pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [3H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [3H]Glu released in a depolarization‐ (induced by 4‐aminopyridine) and Ca2+‐dependent manner. In contrast, fluorescein, the halogen‐free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [3H]Glu uptake, failed to change the amount of exocytotically released [3H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Cholinergic receptor-mediated phosphorylation and activation of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells

Abstract: We have identified a 56‐kilodalton protein in cultured bovine adrenal chromaffin cells that is phos‐phorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one‐ and two‐dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis. Two‐dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+‐dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP‐dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+‐dependent protein kinase. After 2 min phosphorylation by a cyclic AMP‐dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.

Glutamate uptake into synaptic vesicles: competitive inhibition by bromocriptine.

Abstract: The ATP‐dependent uptake of l‐glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the pep‐tide‐containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested; the IC5o was de‐termined to be 22 μM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate trans‐locator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.

Glutamate uptake system in the presynaptic vesicle: Glutamic acid analogs as inhibitors and alternate substrates

A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [3H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (Ki<1mM) was observed fortrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) anderythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki=∼3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity forrans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; alll- anddl-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

Abstract: The ATP‐dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP‐dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP‐dependent glutamate uptake system is reduced by 68% in the staggerer and 57–67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP‐dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.

Accumulated glutamate levels in the synaptic vesicle are not maintained in the absence of active transport

We have investigated factors which may affect accumulated glutamate levels in synaptic vesicles and glutamate efflux. Agents which dissipate the electrochemical proton gradient resulted in a rapid reduction of steady-state vesicular glutamate levels, which was prevented by N-ethylmaleimide. Glutamate efflux was found to occur even in the presence of an electrochemical proton gradient, but was effectively inhibited by N-ethylmaleimide. These results suggest that accumulated glutamate levels in synaptic vesicles are not maintained unless glutamate is taken up continuously by an active transport mechanism, and they could provide an explanation for the lack of convincing evidence for the enrichment of endogenous glutamate in isolated synaptic vesicles.

Ontogeny of glutamate accumulating activity in rat brain synaptic vesicles.

The relationship between the ontogeny of the vesicular glutamate uptake system and synaptogenesis in rats was investigated. For this purpose we have developed a simplified procedure for the preparation of crude synaptic vesicles which are sufficiently pure to demonstrate a highly ATP-dependent glutamate uptake. ATP-dependent glutamate uptake into synaptic vesicles was found to increase dramatically starting on postnatal day 10 and reaching a maximum on day 30 (76 +/- 40 and 657 +/- 40 pmol/mg protein/10 min, respectively), correlating well with the active period of synaptogenesis. Stimulation of uptake by chloride also developed in parallel with the vesicular glutamate uptake. In contrast, combined non-ATP-dependent uptake and non-specific binding remained constant (21 +/- 6 pmol/mg protein/10 min). This development of vesicular glutamate uptake during the period of synaptogenesis supports the notion that synaptic vesicles play an important role in glutamate synaptic transmission.

Synapsin I is associated with cholinergic nerve terminals in the electric organs of Torpedo, Electrophorus, and Malapterurus and copurifies with Torpedo synaptic vesicles.

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.