Magdalena Torres | UCM - Academia.edu (original) (raw)

Papers by Magdalena Torres

Artificial Organs, 2021

High glutamate levels after head trauma or cerebral ischemia have neurotoxic effects. The objecti... more High glutamate levels after head trauma or cerebral ischemia have neurotoxic effects. The objective of the present study was to evaluate the efficacy of hemodialysis to remove glutamate from the blood and to assess the behavior of this small molecule. Ten patients with end‐renal disease on hemodialysis were included in the study. Glutamate clearance was evaluated within the first hour of hemodialysis on a midweek dialysis day on five patients who underwent low flux hemodialysis, whereas the other five patients underwent highly efficient hemodialysis (high flux hemodialysis on one day and online hemodiafiltration on another day). Glutamate clearance with hemodialysis was very effective and did not show any differences between the techniques (low flux: 214 [55], high flux: 204 [37], online hemodiafiltration: 202 [16], median (interquartile range), P = .7). Glutamate clearance was almost equivalent to vascular access plasma flow and it was not affected by dialyzer permeability or ultra...

Cancer research, 1986

The effect of insulin on glucose metabolism through different pathways and the glucose transporte... more The effect of insulin on glucose metabolism through different pathways and the glucose transporters in Harding-Passey melanoma cells have been studied. Glucose was utilized at a rate of 6.9 +/- 2.3 (SD) mumol X g-1 X h-1 with 86% transformed into lactate and pyruvate and only 0.43 and 3% metabolized through the tricarboxylic acid cycle and the pentose phosphate pathway, respectively. Of the total glucose consumed 2% was used in protein synthesis and 2% was used for lipid synthesis. Hexokinase isoenzyme was type I and enolase was present mainly in the alpha gamma hybrid form. The glucose transporters were cytochalasin B sensitive. The number of high affinity cytochalasin B binding sites was 175,000 receptors/cell (about 0.6 pmol/mg protein) and Kd = 1 X 10(-7) M. Insulin increased glucose utilization and lactate production by about 70% and caused a 56% increase in transport without alterations in the Kd of the site. Insulin receptors were quantified by binding assay using 125I-insuli...

Journal of Neurochemistry, 2002

We studied the effect of cultured endothelial cells on the secretion of catecholamines by culture... more We studied the effect of cultured endothelial cells on the secretion of catecholamines by cultured bovine chromaffin cells. Chromaffin cell catecholamine secretion was stimulated by either boluses of potassium (K+) or the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Endothelial cells inhibited the catecholamine release and stimulatory effects of K+ and DMPP. This inhibition increased with time, and in 25 min the initial stimulated secretory response (100%) to 30 mM K+ or 25 microM DMPP dropped to 45 +/- 3% and 53.5 +/- 2.3%, respectively. This endothelial cells-induced inhibition was blocked by the nitric oxide synthase inhibitors N-nitro-L-arginine methyl ester (L-NAME) and N-monoethyl-L-arginine (L-NMMA), and by the guanylate cyclase inhibitor methylene blue, indicating that the L-arginine/nitric oxide/cyclic GMP pathway is involved in this endothelial cell-chromaffin cell interaction. In the absence of endothelial cells, incubation of chromaffin cells with L-NAME, L-NMMA, or methylene blue also augmented the secretagogue-induced catecholamine secretion, indicating that nitric oxide from chromaffin cells could be implicated in an autoinhibitory process of catecholamine release. These results provide indirect evidence for the presence of nitric oxide synthase in bovine adrenomedullary chromaffin cells. Our results show that there is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that an additional level of inhibition is observed when cultured vascular endothelial cells are present. These two inhibitory processes may have different origins, but they appear to converge into a common pathway, the L-arginine/nitric oxide synthase/guanylate cyclase pathway.

British Journal of Pharmacology, 1991

Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents... more Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 + 0.65 x 101 M and Bm.x value of 5420 + 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 + 0.53 x 10-9M and a B... value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2 The diadenosine polyphosphates, Ap3A, Ap4A, ApsA and Ap6A, displaced [3H]-Ap4A from the two binding sites, the K; values being 1.0nm, 0.013nm, 0.013nm and 0.013nm for the very high affinity binding site and 0.5juM, 0.13 pM, 0.062 gm and 0.75 uM for the second binding site. 3 The ATP analogues displaced [3H]-Ap4A with the potency order of the P2,receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-fl-S) > 5'-adenylyl imidodiphosphate (AMP-PNP) > cfl-methylene ATP (a,fl-MeATP), in both binding sites. The Ki values were respectively 0.075 nm, 0.2 nm and 0.75 nm for the very high affinity binding site and 0.125 gM, 0.5 UM and 0.9,UM for the second binding site.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulat... more Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.

Neurobiology of Disease, 2019

In fragile X syndrome, the absence of Fragile X Mental Retardation Protein (FMRP) is known to alt... more In fragile X syndrome, the absence of Fragile X Mental Retardation Protein (FMRP) is known to alter postsynaptic function, although alterations in presynaptic function also occur. We found that the potentiation of glutamate release induced by the β adrenergic receptor (βAR) agonist isoproterenol is absent in cerebrocortical nerve terminals (synaptosomes) from mice lacking FMRP (Fmr1 KO), despite the normal cAMP generation. The glutamate release induced by moderate stimulation of synaptosomes with 5 mM KCl was not potentiated in Fmr1 KO

Cancer Research

Neuroscience Research Communications

The Journal of biological chemistry, Jan 5, 1986

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake... more Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the i...

FEBS Letters, 1991

Di(I ,N~.cth~aoadcnosin©)5 '. 5'".P ,P.tetrapitosphate, ¢.(Ap,A), a fluorescent analog of Ap4A ha... more Di(I ,N~.cth~aoadcnosin©)5 '. 5'".P ,P.tetrapitosphate, ¢.(Ap,A), a fluorescent analog of Ap4A has bc©n synthesized by reaction of 2.¢hloroacetalde. hyde witlt Ap~A. At neutral p H this Ap~A analoll presents d~araeteristie maxima at 26~ and 274 nm, shoulders at ca 260 anti ,310 nnl and moderat~ fluorescence {,~,,, ~107 nm, 2,,, 410 nm), Enzymatic hydrolysis oF the phosphate backbone produced a slight hyperehromic effect but a notorious increase of the fluorescence emiision. Cytosolic ©xtracts fron~ adrenochromaffin tissue as well as cultured chromaltin cells were able to split c(Ap, A) and catabolize the rcsultinj ¢.nucleotide moieties up to c-Ado, Ap,~A; Ap~A fluoctstcnt deriv~ttive; Ap4A splittin8 enzyme; Ectoenzyme; Chromafl~n cell Published by Elsevier Science Publishers B, V.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1984

Methacholine (3 pM) and sodium nitroprusside (300/~M) increased cGMP-dependent protein kinase act... more Methacholine (3 pM) and sodium nitroprusside (300/~M) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2/~M cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0°C) and with an abbreviated incubation time (2.5 min). When assayed at 30°C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1/tM cGMP, or endogenously, by treating intact canine trachealis with methachoUne or sodium nitroprusside. By assaying instead at 0°C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.

Analytical Biochemistry, 1992

The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characteri... more The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.

BMC Neuroscience, 2013

Background: In terms of vesicular recycling, synaptic efficiency is a key determinant of the fide... more Background: In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.

Journal of Cell Science, 2012

Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundame... more Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles. Decreasing the extent of exocytosis dramatically increased the proportion of synaptic boutons that exhibited strong FM1-43-unloading and dramatically reduced the number of endosome-like structures. Hence, we concluded that efficient recycling of synaptic vesicles is concomitant with the formation of non-releasable endosomes in both types of synaptic boutons, although to different extents. Furthermore, cell maturation in culture increased the proportion of synaptic boutons that were capable of an intense release response, whereas the chronic blockage of synaptic activity diminished the capacity of boutons to release dye. by either a weak or a strong loss of FM1-43. Endocytosis in both subsets of nerve terminals was sensitive to the dynamin inhibitor dynasore, which completely prevented dye unloading and dramatically diminished the number of synaptic vesicles. Decreasing the intensity of exocytosis increased the proportion of synaptic boutons that lose large amounts of dye and reduced the presence of endosome-like structures. Together, these findings demonstrate that efficient vesicle-recycling pathways coexist with those pathways that involve the formation of non-releasable endosomes in synaptic boutons, although in different proportions.

From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide ... more From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright a b s t r a c t Phytoestrogens are a group of plant-derived compounds that include mainly isoflavones like daidzein. Phytoestrogens prevent neuronal damage and improve outcome in experimental stroke; however, the mechanisms of this neuroprotective action have not been fully elucidated. In this context, it has been postulated that phytoestrogens might activate the peroxisome proliferator-activated receptor-c (PPARc), which exerts neuroprotective effects in several settings. The aim of this study was to determine whether the phytoestrogen daidzein elicits beneficial actions in neuronal cells by mechanisms involving activation of PPARc. Our results show that daidzein (0.05–5 lM) decreases cell death induced by exposure to oxygen–glucose deprivation (OGD) from rat cortical neurons and that improves synaptic function, in terms of increased synaptic vesicle recycling at nerve terminals, being both effects inhibited by the PPARc antagonist T0070907 (1 lM). In addition, this phytoestrogen activated PPARc in neuronal cultures, as shown by an increase in PPARc transcriptional activity. Interestingly, these effects were not due to binding to the receptor ligand site, as shown by a TR-FRET PPARc competitive binding assay. Conversely, daidzein increased PPARc nuclear protein levels and decreased cytosolic ones, suggesting nuclear trans-location. We have used the receptor antagonist (RE) fulvestrant to study the neuroprotective participation of daidzein via estrogen receptor and at least in our model, we have discarded this pathway. These results demonstrate that the phytoestrogen daidzein has cytoprotective properties in neurons, which are due to an increase in PPARc activity not mediated by direct binding to the receptor ligand-binding domain but likely due to post-translational modifications affecting its subcellular location and not depending to the RE and it is not additive with the agonist rosiglitazone.

Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they med... more Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1a protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals.

Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at ce... more Cross-talk between metabotropic glutamate receptor 7 and beta adrenergic receptor signaling at cerebrocortical nerve terminals, Neuropharmacology (2015),