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1.
J Biol Chem ; 293(28): 11179-11194, 2018 07 13.
Article in English | MEDLINE | ID: mdl-29802198

ABSTRACT

Striatin-1, a subunit of the serine/threonine phosphatase PP2A, is preferentially expressed in neurons in the striatum. As a member of the striatin family of B subunits, striatin-1 is a core component together with PP2A of a multiprotein complex called STRIPAK, the striatin-interacting phosphatase and kinase complex. Little is known about the function of striatin-1 or the STRIPAK complex in the mammalian striatum. Here, we identify a selective role for striatin-1 in striatal neuron maturation. Using a small hairpin RNA (shRNA) knockdown approach in primary striatal neuronal cultures, we determined that reduced expression of striatin-1 results in increased dendritic complexity and an increased density of dendritic spines, classified as stubby spines. The dendritic phenotype was rescued by co-expression of a striatin-1 mutant construct insensitive to the knockdown shRNA but was not rescued by co-expression of PP2A- or Mob3-binding deficient striatin-1 constructs. Reduction of striatin-1 did not result in deficits in neuronal connectivity in this knockdown model, as we observed no abnormalities in synapse formation or in spontaneous excitatory postsynaptic currents. Thus, this study suggests that striatin-1 is a regulator of neuronal development in striatal neurons.


Subject(s)
Calmodulin-Binding Proteins/metabolism , Membrane Proteins/metabolism , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , Protein Phosphatase 2/metabolism , Spine/cytology , Spine/metabolism , Animals , Calmodulin-Binding Proteins/genetics , Cells, Cultured , Female , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Multiprotein Complexes/genetics , Nerve Tissue Proteins/genetics , Neuronal Plasticity , Neurons/metabolism , Protein Phosphatase 2/genetics , Protein Subunits , Rats , Rats, Sprague-Dawley
2.
Elife ; 62017 06 14.
Article in English | MEDLINE | ID: mdl-28613156

ABSTRACT

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Gene Expression Regulation , Microtubule-Associated Proteins/metabolism , Phosphoproteins/metabolism , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , HEK293 Cells , Humans
3.
J Neurosci ; 37(10): 2709-2722, 2017 03 08.
Article in English | MEDLINE | ID: mdl-28167675

ABSTRACT

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling.SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We find that the protein ARPP-16, which is highly expressed in striatal medium spiny neurons, acts as a selective inhibitor of certain forms of the serine/threonine protein phosphatase, PP2A, when phosphorylated by the kinase, MAST3. Under basal conditions, ARPP-16 is phosphorylated by MAST3 to a very high stoichiometry. However, the actions of MAST3 are antagonized by dopamine and cAMP-regulated signaling leading to disinhibition of ARPP-16 and increased PP2A action.


Subject(s)
Corpus Striatum/metabolism , Gene Expression Regulation, Enzymologic/physiology , Microtubule-Associated Proteins/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Phosphorylation
4.
J Biol Chem ; 292(4): 1462-1476, 2017 01 27.
Article in English | MEDLINE | ID: mdl-27998980

ABSTRACT

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.


Subject(s)
Cell Nucleus/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Dopamine/metabolism , Receptors, Dopamine D1/metabolism , Signal Transduction/physiology , Animals , Cell Nucleus/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Dopamine/genetics , Dopamine and cAMP-Regulated Phosphoprotein 32/genetics , Male , Mice , Phosphorylation/physiology , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Receptors, Dopamine D1/genetics
5.
PLoS One ; 7(3): e32180, 2012.
Article in English | MEDLINE | ID: mdl-22461884

ABSTRACT

"Hypomyelination and Congenital Cataract", HCC (MIM #610532), is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS). Using heterozygous mice expressing the b-galactosidase (LacZ) gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10) and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC)/Dorsal Root Ganglion (DRG) sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination.


Subject(s)
Cataract/genetics , Hereditary Central Nervous System Demyelinating Diseases/genetics , Mutation , Neurons/metabolism , Oncogene Proteins/genetics , Animals , Animals, Newborn , Blotting, Western , Brain/cytology , Brain/growth & development , Brain/metabolism , Cataract/congenital , Female , Gene Expression Regulation, Developmental , HeLa Cells , Humans , In Situ Hybridization , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Neurons/cytology , Oncogene Proteins/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sciatic Nerve/growth & development , Sciatic Nerve/metabolism , Sciatic Nerve/ultrastructure , Time Factors , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
6.
J Neurochem ; 117(3): 516-27, 2011 May.
Article in English | MEDLINE | ID: mdl-21348870

ABSTRACT

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors.


Subject(s)
Glycine Plasma Membrane Transport Proteins/metabolism , Magnesium/pharmacology , Presynaptic Terminals/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Synaptosomes/drug effects , Animals , Aspartic Acid/metabolism , Calcium/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Glutamic Acid/metabolism , Glycine/metabolism , Hippocampus/ultrastructure , Immunoprecipitation/methods , Male , N-Methylaspartate/metabolism , Pipecolic Acids/pharmacology , Piperidines/pharmacology , Presynaptic Terminals/metabolism , Rats , Rats, Sprague-Dawley , Tetrahydronaphthalenes/pharmacology , Tritium/metabolism
7.
Cereb Cortex ; 20(8): 1974-84, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20034999

ABSTRACT

Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.


Subject(s)
Down-Regulation/physiology , Exocytosis/physiology , Glutamic Acid/metabolism , Neocortex/metabolism , Presynaptic Terminals/metabolism , Up-Regulation/physiology , gamma-Aminobutyric Acid/metabolism , tat Gene Products, Human Immunodeficiency Virus/physiology , Adult , Aged , Animals , Excitatory Amino Acid Antagonists/pharmacology , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Neocortex/cytology , Organ Culture Techniques , Peptide Fragments/pharmacology , Potassium/toxicity , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Synaptosomes/metabolism , Young Adult
8.
J Neurosci ; 28(47): 12231-40, 2008 Nov 19.
Article in English | MEDLINE | ID: mdl-19020017

ABSTRACT

The effects of the recombinant chemokine human RANTES (hRANTES) on the release of glutamate from human neocortex glutamatergic nerve endings were investigated. hRANTES facilitated the spontaneous release of d [(3)H]D-aspartate ([(3)H]DASP-) by binding Pertussis toxin-sensitive G-protein-coupled receptors (GPCRs), whose activation caused Ca(2+) mobilization from inositol trisphosphate-sensitive stores and cytosolic tyrosine kinase-mediated phosphorylations. Facilitation of release switched to inhibition when the effects of hRANTES on the 12 mM K(+)-evoked [(3)H]D-ASP exocytosis were studied. Inhibition of exocytosis relied on activation of Pertussis toxin-sensitive GPCRs negatively coupled to adenylyl cyclase. Both hRANTES effects were prevented by met-RANTES, an antagonist at the chemokine receptors (CCRs) of the CCR1, CCR3, and CCR5 subtypes. Interestingly, human neocortex glutamatergic nerve endings seem to possess all three receptor subtypes. Blockade of CCR1 and CCR5 by antibodies against the extracellular domain of CCRs prevented both the hRANTES effect on [(3)H]D-ASP release, whereas blockade of CCR3 prevented inhibition, but not facilitation, of release. The effects of RANTES on the spontaneous and the evoked release of [(3)H]D-ASP were also observed in experiments with mouse cortical synaptosomes, which may therefore represent an appropriate animal model to study RANTES-induced effects on neurotransmission. It is concluded that glutamate transmission can be modulated in opposite directions by RANTES acting at distinct CCR receptor subtypes coupled to different transduction pathways, consistent with the multiple and sometimes contrasting effects of the chemokine.


Subject(s)
Chemokine CCL5/pharmacology , Glutamic Acid/metabolism , Neocortex/drug effects , Neocortex/metabolism , Adult , Aged , Analysis of Variance , Animals , Aspartic Acid/pharmacology , Calcium/metabolism , Chemokine CCL5/antagonists & inhibitors , D-Aspartic Acid/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Humans , In Vitro Techniques , Macrocyclic Compounds/pharmacology , Male , Mice , Middle Aged , Oxazoles/pharmacology , Receptors, CCR/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , Time Factors , Tritium/metabolism , Young Adult
9.
Neuropharmacology ; 55(4): 474-82, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18625255

ABSTRACT

The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [3H]d-aspartate ([3H]D-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1-100microM) potentiated the K+(12mM)-evoked [3H]D-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [3H]D-ASP exocytosis when applied at 0.3microM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30-100microM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [3H]D-ASP exocytosis caused by 0.3microM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50microM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3microM 3,5-DHPG failed to facilitate the K+-evoked [3H]D-ASP overflow from mGlu5 receptor knockout (mGlu5-/-) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50microM 3,5-DHPG failed to affect the [3H]D-ASP exocytosis from cortical synaptosomes obtained from crv4/crv4 and mGlu5-/-mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the "high affinity" binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the "low affinity" binding sites.


Subject(s)
Cerebral Cortex/cytology , Exocytosis/physiology , Glutamic Acid/metabolism , Presynaptic Terminals/physiology , Receptors, Metabotropic Glutamate/physiology , Animals , Aspartic Acid/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Excitatory Amino Acid Agents/pharmacology , Exocytosis/drug effects , Gene Expression/physiology , Glycine/analogs & derivatives , Glycine/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Potassium Chloride/pharmacology , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/deficiency , Resorcinols/pharmacology , Synaptosomes/drug effects , Synaptosomes/metabolism , Syntaxin 1/metabolism , Tritium/metabolism , Vesicular Glutamate Transport Protein 1/metabolism
10.
Int Rev Neurobiol ; 82: 339-56, 2007.
Article in English | MEDLINE | ID: mdl-17678970

ABSTRACT

Human immunodeficiency virus 1 (HIV-1)-associated dementia (HAD) represents a common complication of HIV-1 infection. Antiretroviral therapy has diminished its incidence, but it is insufficient to eradicate the problem. HAD depends on the presence of the virus in central nervous system (CNS), but the molecular mechanisms involved are not completely understood. It is widely accepted that proteins shed by the virus, such as the envelope glycoprotein gp120 and the nonstructural viral protein Tat, may themselves cause alterations to CNS. By one side, viral proteins are toxic to neurons because of their ability (1) to act as excitotoxins and (2) to evoke the release of endogenous neurotoxins and/or proinflammatory cytokines. By the other side, evidences are emerging that viral components can alter neuronal functions either by modifying the release of neurotransmitters or by influencing the functions of classical receptors controlling central neurotransmission. We here review some results concerning the effects of Tat on cholinergic and noradrenergic neurotransmission in human and rat cortex. The protein can induce the release of acetylcholine from both human and rat cortical cholinergic nerve terminals in a specie-specific manner. In human cholinergic terminals, Tat-mediated releasing effect depends on activation of receptors belonging to I group of metabotropic glutamate receptors (mGluRs), while in rat terminals Tat-induced effect involves the activation of a so far unknown receptor. The protein, unable on its own to release noradrenaline from human and rat cortical noradrenergic nerve endings, potentiates the release of amine induced by presynaptic NMDA receptors. Also in this case, Tat effect involves activation of a receptor belonging to the group I mGluRs, in particular of the mGluR1 subtype. The finding that group I mGluRs may represent a preferential target of the protein in CNS may be relevant to the proposal of new therapeutic approaches for the cure of HAD.


Subject(s)
Gene Products, tat/pharmacology , HIV-1/metabolism , Receptors, Metabotropic Glutamate/physiology , Synaptic Transmission/physiology , Animals , Central Nervous System/drug effects , Central Nervous System/metabolism , Gene Products, tat/genetics , Humans , Neurotransmitter Agents/metabolism , Receptors, Metabotropic Glutamate/drug effects , Receptors, Presynaptic/drug effects , Synaptic Transmission/drug effects , tat Gene Products, Human Immunodeficiency Virus
11.
J Neurosci Res ; 85(16): 3657-65, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17671992

ABSTRACT

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.


Subject(s)
Exocytosis/physiology , Glutamic Acid/metabolism , Hippocampus/metabolism , Presynaptic Terminals/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Vesicular Glutamate Transport Proteins/metabolism , Animals , Aspartic Acid/metabolism , Aspartic Acid/pharmacology , Autoreceptors/drug effects , Autoreceptors/physiology , Dose-Response Relationship, Drug , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Exocytosis/drug effects , Glycine/pharmacology , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp120/pharmacology , Hippocampus/drug effects , Male , N-Methylaspartate/pharmacology , Presynaptic Terminals/drug effects , Rats , Receptors, N-Methyl-D-Aspartate/agonists , Receptors, N-Methyl-D-Aspartate/drug effects , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Synaptosomes , Vesicular Glutamate Transport Proteins/drug effects
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