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Galo Ramirez - One of the best experts on this subject based on the ideXlab platform.
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Kainate-Triggered Currents in Xenopus Oocytes Injected with Chick Retinal Membrane Fragments: Effect of Guanine Nucleotides
2020Co-Authors: Javier S Burgos, Ana Barat, Jordi Aleu, Carles Solsona, Jordi Marsal, Galo RamirezAbstract:PURPOSE. To electrophysiologically characterize ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection. METHODS. A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at Ϫ70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining. RESULTS. Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% Ϯ 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 Ϯ 72.3 m 2 ). Bath-applied kainate induced dose-dependent (EC 50 : 64 Ϯ 7 M), nondesensitizing inward currents (15-90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by ␥-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine Nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner. CONCLUSIONS. An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of Guanine Nucleotides toward glutamate receptors and their potential role as neuropro- These results add to the accumulated evidence on the antagonistic behavior of Guanine Nucleotides (GNs) at ionotropic glutamate receptors, in very diverse experimental setups, including agonist displacement, electrophysiological recording and neuroprotection paradigms. 21,22 MATERIALS AND METHODS Chick Retinal Membrane Preparation All experiments with animals (chicks, Xenopus, and rabbits) followed our institutional guidelines for care and handling of laboratory animals, in full agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six-day-old white leghorn chicks were used as the source of retinal membrane fragments. Lysed membranes, prepared as described, 7 were resuspended in 10 mM HEPES (pH 7.4), containing 140 mM KCl and 20 mM NaCl, at a protein concentration of 2 mg/mL, stored in liquid nitrogen, and thawed and sonicated for 10 seconds in an ice water bath just before injection. A control solution without membranes was similarly processed and used for sham-injected control chicks. Oocyte Preparation and Injection Mature female Xenopus laevis were obtained from the Centre d'Elevage des Xénopes, CRBM (Montpellier, France), and kept in chlorine-free fresh water, at 22°C. Discrete ovary portions were removed from anesthetized frogs 23 and stage-V/VI oocytes 24 were individually dissected and kept, at 15°C to 17°C, in sterile modified Barth's solution (10 mM HEPES [pH 7.4], 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , and 2.4 mM NaHCO 3 ) supplemented with penicillin (100 IU/mL) and streptomycin (0.1 mg/mL). Oocytes were further treated with collagenase (clostridiopeptidase A: EC3.4.24.3; type IA, 0.5 mg/mL; Sigma-Aldrich, St. Louis, MO), for 50 minutes at room temperature, to remove enveloping cells. 25 From th
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Guanine Nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation
FEBS Letters, 1998Co-Authors: Javier S Burgos, Diogo O Souza, Ana Barat, Galo RamirezAbstract:Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that Guanine Nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of Guanine Nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.
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Effects of Guanine Nucleotides on Glutamate-Induced Chemiluminescence in Rat Hippocampal Slices Submitted to Hypoxia
Neurochemical Research, 1998Co-Authors: Andrea Regner, Galo Ramirez, Adriane Belló-klein, Diogo SouzaAbstract:Glutamate significantly increased levels of spontaneous chemiluminescence (CL) in rat hippocampal slices incubated under hypoxic conditions. Although it has been previously shown that Guanine Nucleotides (GN) displace glutamate from several of its receptors, in our study only GMP, as well as the glutamate antagonist MK-801, was able to reverse the increase in CL provoked by glutamate. On the other hand, not only GTP or Gpp(NH)p failed to reverse the action of glutamate, but they increased CL production like glutamate. This effect of GTP/Gpp(NH)p was also reversed by GMP. We concluded that, under neurotoxic conditions, GMP acted as an antagonist and GTP or Gpp(NH)p acted as agonists of glutamate. These results reinforced the evidence of the existence of extracellular site(s) for GN and indicated a possible role for GN in excitotoxicity.
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Guanine Nucleotides inhibit the stimulation of gfap phosphorylation by glutamate
Neuroreport, 1995Co-Authors: Carla I Tasca, Susana Tchernin Wofchuk, Diogo O Souza, Galo Ramirez, Richard RodnightAbstract:Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by Guanine Nucleotides. Recent work has shown that Guanine Nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that Guanine Nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.
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differential effects of Guanine Nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum
FEBS Letters, 1994Co-Authors: Murilo Monteiro Paz, Galo Ramirez, Milagros Ramos, Diogo O SouzaAbstract:In G protein-coupled receptors, neurotransmitter-induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor—G protein complex. In the present study we show that, in the chick optic tectum, Guanine Nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP-PNP, a non-hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP-binding sites are pre-saturated with GMP-PNP. Experiments carried out with these membranes show that GMP-PNP, GDP-S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP-PNP activates adenylate cyclase activity by binding to G proteins.
Diogo O Souza - One of the best experts on this subject based on the ideXlab platform.
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Guanine Nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation
FEBS Letters, 1998Co-Authors: Javier S Burgos, Diogo O Souza, Ana Barat, Galo RamirezAbstract:Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that Guanine Nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of Guanine Nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.
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Guanine Nucleotides inhibit the stimulation of gfap phosphorylation by glutamate
Neuroreport, 1995Co-Authors: Carla I Tasca, Susana Tchernin Wofchuk, Diogo O Souza, Galo Ramirez, Richard RodnightAbstract:Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by Guanine Nucleotides. Recent work has shown that Guanine Nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that Guanine Nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.
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differential effects of Guanine Nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum
FEBS Letters, 1994Co-Authors: Murilo Monteiro Paz, Galo Ramirez, Milagros Ramos, Diogo O SouzaAbstract:In G protein-coupled receptors, neurotransmitter-induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor—G protein complex. In the present study we show that, in the chick optic tectum, Guanine Nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP-PNP, a non-hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP-binding sites are pre-saturated with GMP-PNP. Experiments carried out with these membranes show that GMP-PNP, GDP-S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP-PNP activates adenylate cyclase activity by binding to G proteins.
Lelio Orci - One of the best experts on this subject based on the ideXlab platform.
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the amino terminus of adp ribosylation factor arf is a critical determinant of arf activities and is a potent and specific inhibitor of protein transport
Journal of Biological Chemistry, 1992Co-Authors: Richard A Kahn, Paul A Randazzo, Tito Serafini, O Weiss, C Rulka, Jenny Clark, Mylene Amherdt, Peter P Roller, Lelio OrciAbstract:Deletion of the amino-terminal 17 residues from human ADP-ribosylation factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind Guanine Nucleotides with high affinity. Unlike the wild type, the binding of Guanine Nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.
Hsiuying T Yang - One of the best experts on this subject based on the ideXlab platform.
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modulation of neuropeptide ff receptors by Guanine Nucleotides and cations in membranes of rat brain and spinal cord
Journal of Neurochemistry, 1993Co-Authors: Kemal Payza, Hsiuying T YangAbstract:Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF (NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I-YLFQPQRFamide (125I-Y8Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine Nucleotides dose-dependently inhibited specific 125I-Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 +/- 6 and 71 +/- 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP > or = GDP >> GMP, ATP. The Guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 microM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-O-(3-thiotriphosphate). Our data suggest that 125I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.
Claudia N. Tomes - One of the best experts on this subject based on the ideXlab platform.
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Membrane-permeable Rab27A is a regulator of the acrosome reaction: Role of geranylgeranylation and Guanine Nucleotides
Cellular signalling, 2018Co-Authors: Matías A. Bustos, Ornella Lucchesi, María Celeste Ruete, Claudia N. TomesAbstract:Abstract The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and Guanine Nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized — but not in non-permeabilized — cells when geranylgeranylated and active. When GDP-β-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-β-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.
